CN106148362B - Novel coelenterazine substrates and methods of use thereof - Google Patents

Abstract

An isolated polynucleotide encoding a modified luciferase polypeptide and a substrate. The OgLuc variant polypeptide has at least 60% amino acid sequence identity to SEQ ID No. 1 and has at least one amino acid substitution at a position corresponding to an amino acid in SEQ ID No. 1. The OgLuc variant polypeptide has at least one of enhanced luminescence, enhanced signal stability, and enhanced protein stability relative to a corresponding polypeptide of a wild-type oplophorus luciferase.

Description

Novel coelenterazine substrates and methods of use thereof

Cross Reference to Related Applications

This application claims priority to U.S. provisional application No. 61/409,422, filed on 2/11/2010, which is incorporated herein by reference in its entirety.

Background

Luciferase secreted from the deep sea shrimp Cereus gracilis (Oplophorus gracilirostris) has been shown to possess many interesting features, such as high activity, high quantum yield and broad substrate specificity (including, for example, coelenterazine and various coelenterazine analogs). When the oxidation of coelenterazine (substrate) with molecular oxygen is catalyzed by Oplophorus luciferase, a bioluminescent reaction of Oplophorus occurs, resulting in light of maximum intensity at 462nm and product CO ₂And coelenteramide (Shimomura et al, Biochemistry,17:994 (1978)). The optimal luminescence occurs at 40 ℃ in the presence of 0.05-0.1M NaCl at pH 9 and, due to the unique resistance of the enzyme to heat, visible luminescence occurs at temperatures above 50 ℃ when high purity enzymes are used or above 70 ℃ when partially purified enzymes are used. At pH 8.7, Shimomura et al (1978) reported that native luciferase has a molecular weight of about 130kDa, apparently comprising 4 monomers of 31kDa each; at lower pH, the native luciferase tends to polymerize.

Recent studies have shown that oplophorus gracilis luciferase is a complex of 35kDa and 19kDa native proteins, i.e. a heterotetramer consisting of two 19kDa components and two 35kDa components. Inouye et al (2000) reported molecular cloning of cDNAs encoding 35kDa and 19kDa proteins of Oplophorus luciferase, and identification of protein components that catalyze luminescence reactions. The cDNA encoding the protein was expressed in bacterial and mammalian cells, and a 19kDa protein was identified as a component capable of catalyzing the luminescent oxidation reaction of coelenterazine (Inouye et al, 2000).

The 19kDa protein of Oplophorus luciferase (GenBank accession BAB13776,196 amino acids) appears to be the smallest catalytic component with luciferase function and its primary structure has no significant similarity to any reported luciferase including imidazopyrazinone luciferase (Lorenz et al, PNAS USA, 88:4438 (1991); Thompson et al, PNAS USA, 86:6567 (1989)). Expression of the 19kDa Protein in E.coli (E.coli) results in the formation of inclusion bodies (Inouye and Sasaki, Protein Expression and Purification,56:261-268 (2007)). The formation of inclusion bodies may be due to protein instability.

The substrate specificity of Acridium luciferases is unexpectedly broad (Inouye and Shimomura. BBRC 223:349 (1997)). For example, dideoxycoelenterazine (i.e., coelenterazine-hh), a coelenterazine analog, is an excellent substrate for Oplophorus luciferase comparable to coelenterazine (Nakamura et al, Tetrahedron, Lett., 38:6405 (1997)). Also, Echinacea luciferases are secreted enzymes, similar to those of the sea-related Schizophyllum commune (Cypridina (Vargula) hilgendorfii) (Johnson and Shimomura, meth.enzyme, 57:331(1978)), which also use imidazopyrazinone type luciferases to emit light.

SUMMARY

In one aspect, the disclosure relates to compounds of formula (Ia) or (Ib):

wherein R is²Selected from the group consisting of:

or C_2-5A linear alkyl group;

R⁶selected from the group consisting of-H, -OH, -NH₂-OC (O) R or-OCH₂OC (O) R;

R⁸selected from the group consisting of:

h or lower cycloalkyl;

wherein R is³And R⁴Are both H or both C_1-2An alkyl group;

w is-NH₂Halogen substituted、-OH、-NHC(O)R、-CO₂R；

X is-S-, -O-or-NR²²-；

Y is-H, -OH OR-OR¹¹；

Z is-CH-or-N-;

each R¹¹Independently is-C (O) R' or-CH₂OC(O)R”；

R²²Is H, CH₃Or CH₂CH₃；

Each R is independently C_1-7Straight chain alkyl or C_1-7A branched alkyl group;

R' is C_1-7Straight chain alkyl or C_1-7A branched alkyl group;

the bond of the dotted line indicates the presence of an optional ring, which may be saturated or unsaturated;

provided that when R is²Is composed of

When R is⁸Is not that

Provided that when R is²Is composed of

When R is⁸Is that

Or lower cycloalkyl; and

provided that when R is⁶Is NH₂When R is²In order to realize the purpose,

or C_2-5An alkyl group;

or R⁸Is not that

In another aspect, the disclosure relates to a compound selected from the group consisting of:

in one aspect, the disclosure relates to compounds of the formula:

in one aspect, the disclosure relates to an isolated polynucleotide encoding an OgLuc variant polypeptide having at least 60% amino acid sequence identity to SEQ ID No. 1, comprising at least one amino acid substitution at a position corresponding to an amino acid in SEQ ID No. 1, wherein the OgLuc variant polypeptide has enhanced luminescence.

In one aspect, the disclosure relates to an isolated polynucleotide encoding an OgLuc variant polypeptide having at least 60% amino acid sequence identity to SEQ ID No. 1, the OgLuc variant polypeptide including at least one amino acid substitution at a position corresponding to an amino acid in SEQ ID No. 1, wherein the OgLuc variant polypeptide has enhanced luminescence relative to the OgLuc polypeptide of SEQ ID No. 3, with the proviso that the polypeptide encoded by the polynucleotide is not one of those listed in table 47.

In one aspect, the disclosure relates to an isolated polynucleotide encoding an OgLuc variant polypeptide having at least 60% amino acid sequence identity to SEQ ID No. 1, the OgLuc variant polypeptide including at least one amino acid substitution at a position corresponding to an amino acid in SEQ ID No. 1, wherein the OgLuc variant polypeptide has enhanced luminescence relative to the polypeptide of SEQ ID No. 31, with the proviso that the polypeptide encoded by the polynucleotide is not SEQ ID No. 3 or 15.

In one aspect, the disclosure relates to an isolated polynucleotide encoding an OgLuc variant polypeptide having at least 60% amino acid sequence identity to SEQ ID No. 1, the OgLuc variant polypeptide including at least one amino acid substitution at a position corresponding to an amino acid in SEQ ID No. 1, wherein the OgLuc variant polypeptide has enhanced luminescence relative to the polypeptide of SEQ ID No. 29, with the proviso that the polypeptide encoded by the polynucleotide is not SEQ ID No. 3 or 15.

In one aspect, the disclosure relates to an isolated polynucleotide encoding an OgLuc variant polypeptide having at least 80% amino acid sequence identity to the OgLuc polypeptide of SEQ ID No. 1, the OgLuc variant polypeptide comprising the amino acid substitutions A4E, Q11R, a33K, V44I, P115E, Q124K, Y138I, N166R, I90V, F54I, Q18L, F68Y, L72Q, and M75K corresponding to SEQ ID No. 1, and the OgLuc variant polypeptide having luciferase activity.

In one aspect, the disclosure relates to a polypeptide encoding a polypeptide corresponding to SEQ ID NO:1, wherein an OgLuc variant polypeptide having at least 80% amino acid sequence identity to an OgLuc polypeptide of SEQ ID NO:1 is glutamic acid at the 4 th amino acid, arginine at the 11 th amino acid, leucine at the 18 th amino acid, lysine at the 33 th amino acid, isoleucine at the 44 th amino acid, isoleucine at the 54 th amino acid, tyrosine at the 68 th amino acid, glutamine at the 72 th amino acid, lysine at the 75 th amino acid, valine at the 90 th amino acid, glutamic acid at the 115 th amino acid, lysine at the 124 th amino acid, isoleucine at the 138 th amino acid, and arginine at the 166 th amino acid, and the OgLuc variant polypeptide has luciferase activity.

In one aspect, the disclosure relates to an isolated polynucleotide encoding an OgLuc variant polypeptide having at least 80% amino acid sequence identity to the OgLuc polypeptide of SEQ ID No. 1, the OgLuc variant polypeptide comprising the amino acid substitutions A4E, Q11R, a33K, V44I, P115E, Q124K, Y138I, N166R, Q18L, F54I, L92H, and Y109F corresponding to SEQ ID No. 1, and the OgLuc variant polypeptide having luciferase activity.

In one aspect, the disclosure relates to an isolated polynucleotide encoding an OgLuc variant polypeptide having at least 80% amino acid sequence identity to the OgLuc polypeptide of SEQ ID No. 1, the OgLuc variant polypeptide comprising the amino acid substitutions A4E, Q11R, a33K, V44I, a54I, F77Y, I90V, P115E, Q124K, Y138I, and N166R corresponding to SEQ ID No. 1, and the OgLuc variant polypeptide having luciferase activity.

In one aspect, the disclosure relates to an isolated polynucleotide encoding an OgLuc variant polypeptide having at least 80% amino acid sequence identity to the OgLuc polypeptide of SEQ ID No. 1, wherein the amino acid at position 4 corresponding to SEQ ID No. 1 is glutamic acid, the amino acid at position 11 is arginine, the amino acid at position 18 is leucine, the amino acid at position 33 is lysine, the amino acid at position 44 is isoleucine, the amino acid at position 54 is isoleucine, the amino acid at position 92 is histidine, the amino acid at position 109 is phenylalanine, the amino acid at position 115 is glutamic acid, the amino acid at position 124 is lysine, the amino acid at position 138 is isoleucine, and the amino acid at position 166 is arginine, and the OgLuc variant polypeptide has luciferase activity.

In one aspect, the disclosure relates to an isolated polynucleotide encoding an OgLuc variant polypeptide having at least 80% amino acid sequence identity to the OgLuc polypeptide of SEQ ID No. 1, wherein the amino acid at position 4 corresponding to SEQ ID No. 1 is glutamic acid, the amino acid at position 11 is arginine, the amino acid at position 33 is lysine, the amino acid at position 44 is isoleucine, the amino acid at position 54 is isoleucine, the amino acid at position 77 is tyrosine, the amino acid at position 90 is valine, the amino acid at position 115 is glutamic acid, the amino acid at position 124 is lysine, the amino acid at position 138 is isoleucine, and the amino acid at position 166 is arginine, and the OgLuc variant polypeptide has luciferase activity.

In one aspect, the disclosure relates to an isolated polynucleotide comprising a polynucleotide encoding the polypeptide of SEQ ID NO 19.

In one aspect, the disclosure relates to isolated polynucleotides comprising the polynucleotides of SEQ ID NO 18, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 42, SEQ ID NO 88, or SEQ ID NO 92.

In one aspect, the disclosure relates to an isolated polynucleotide encoding a decapod luciferase polypeptide having at least 30% amino acid sequence identity to SEQ ID No. 1, the polypeptide comprising a sequence pattern corresponding to the sequence pattern of formula (VII) and comprising NO more than 5 differences, wherein differences comprise differences according to the OgLuc pattern listed in table 4 relative to mode position 1, 2, 3, 5, 8, 10, 12, 14, 15, 17, or 18 of formula (VII), and a gap or insertion between any mode position of formula (VII) according to the OgLuc pattern listed in table 4, wherein the decapod luciferase produces luminescence in the presence of coelenterazine.

In one aspect, the disclosure relates to a synthetic nucleotide sequence encoding an OgLuc variant polypeptide, the OgLuc variant polypeptide includes a polypeptide having an amino acid sequence substantially similar to that of SEQ ID NO:2 and has 80% or less nucleic acid sequence identity to the parent nucleic acid sequence of SEQ ID NO: 22. SEQ ID NO: 23. SEQ ID NO:24 or SEQ ID NO:25 or a complement thereof having a nucleotide sequence identity of 90% or greater, wherein the reduced sequence identity is a result of codons in the synthetic nucleotide sequence that differ relative to codons in the parent nucleic acid sequence, wherein the synthetic nucleotide sequence encodes an OgLuc variant having at least 85% amino acid sequence identity to a corresponding luciferase encoded by the parent nucleic acid sequence, and wherein the synthetic nucleotide sequence has a reduced number of regulatory sequences relative to the parent nucleic acid sequence.

In one aspect, the disclosure relates to a synthetic nucleotide sequence encoding an OgLuc variant polypeptide, the OgLuc variant polypeptide includes a polypeptide having an amino acid sequence substantially similar to that of SEQ ID NO:14 and has 80% or less nucleic acid sequence identity to the parent nucleic acid sequence of SEQ ID NO:22 or SEQ ID NO:23 or a complement thereof having a nucleic acid sequence identity of at least 90% or more, wherein the reduced sequence identity is a result of codons in the synthetic nucleotide sequence that differ relative to codons in the parent nucleic acid sequence, wherein the synthetic nucleotide sequence encodes a firefly luciferase having at least 85% amino acid sequence identity to the corresponding luciferin enzyme encoded by the parent nucleic acid sequence, and wherein the synthetic nucleotide sequence has a reduced number of regulatory sequences relative to the parent nucleic acid sequence.

In one aspect, the disclosure relates to a synthetic nucleotide sequence encoding an OgLuc variant polypeptide, the OgLuc variant polypeptide includes a polypeptide having an amino acid sequence substantially similar to that of SEQ ID NO:18 and has 80% or less nucleic acid sequence identity to a parent nucleic acid sequence of SEQ ID NO:24 or SEQ ID NO:25 or a complement thereof having a nucleic acid sequence identity of 90% or greater of at least 100 nucleotides, wherein the reduced sequence identity is a result of codons in the synthetic nucleotide sequence that differ relative to codons in the parent nucleic acid sequence, wherein the synthetic nucleotide sequence encodes an OgLuc variant having at least 85% amino acid sequence identity to a corresponding luciferase encoded by the parent nucleic acid sequence, and wherein the synthetic nucleotide sequence has a reduced number of regulatory sequences relative to the parent nucleic acid sequence.

In one aspect, the disclosure relates to a fusion peptide from a signal peptide of Oplophorus gracilis fused to a heterologous protein, wherein the signal peptide is SEQ ID NO:54, wherein the fusion peptide is expressed in and secreted from a cell.

In one aspect, the disclosure relates to a method of generating a polynucleotide encoding an OgLuc variant polypeptide, the method comprising: (a) generating a library of variant fusion proteins using a parent fusion protein construct comprising a parent OgLuc polypeptide and at least one heterologous polypeptide; and (b) screening the library for at least one of enhanced luminescence, enhanced enzyme stability, or enhanced biocompatibility relative to the parent fusion protein construct.

In one aspect, the disclosure relates to a method of generating a codon-optimized polynucleotide encoding a luciferase for an organism, the method comprising: for each amino acid in the luciferase, codons are randomly selected from the two most commonly used codons used in the organism to encode the amino acid to produce a first codon-optimized polynucleotide.

Other aspects of the invention will become apparent by consideration of the detailed description and accompanying drawings.

Brief Description of Drawings

FIG. 1 shows the chemical structures of native coelenterazine, known bis-coelenterazine (coelenterazine-hh), and known coelenterazine-h, where R2, R6, and R8 represent regions of the molecule being modified.

FIG. 2 shows the chemical structures of novel coelenterazine PBI-3939, PBI-3889, PBI-3945, PBI-4002, PBI-3841, PBI-3897, PBI-3896, PBI-3925, PBI-3894, PBI-3932 and PBI-3840.

FIG. 3 shows Km determination for PBI-3939.

FIG. 4 shows the chemical structures of various novel coelenterazines of the present invention.

FIGS. 5A-G show luminescence (RLU) generated from lysed C1+ A4E-expressing bacterial cells using native, known and novel coelenterazine as a substrate. Figures 5A, 5C-5G show independent experiments measuring the luminescence in RLU generated by C1+4AE for known and novel coelenterazine using native coelenterazine as a comparison. Figure 5B shows a fold decrease in luminescence generated by C1+4AE using the substrate shown in figure 5A compared to native coelenterazine.

FIGS. 6A-D show luminescence generated from lysed bacteria expressing different OgLuc variants using native coelenterazine ("Coelente"), known coelenterazine-h ("h, h"), known 2-methyl coelenterazine ("2-me"), known coelenterazine-v ("v"), and novel coelenterazine PBI-3840, PBI-3897, PBI-3889, PBI-3899, PBI-3900, PBI-3912, PBI-3913, PBI-3925, PBI-3897, PBI-3899, PBI-3889, PBI-3939, PBI-3933, PBI-3932, PBI-3946, PBI-3897, PBI-3841, PBI-3896, PBI-3925, and PBI-3945 as substrates.

Figure 7 shows amino acid substitutions in different OgLuc variants.

FIGS. 8A-B show luminescence generated from lysed bacterial cells expressing the OgLuc variant listed in FIG. 7 using as substrates native Coelenterazine ("Coelenterazine"), known Coelenterazine-H ("H"), known Coelenterazine-hh ("H, H"), and novel Coelenterazine PBI-3840, PBI-3925, PBI-3912, PBI-3889, PBI-3939, PBI-3933, PBI-3932, PBI-3946, PBI-3941, and PBI-3896.

FIG. 9 shows luminescence generated from lysed bacterial cells expressing different OgLuc variants using native coelenterazine ("coelenterazine"), known coelenterazine-hh ("h, h"), and novel coelenterazine PBI-3939, PBI-3945, PBI-3840, PBI-3932, PBI-3925, PBI-9894, and PBI-3896 as substrates.

Figure 10 shows amino acid substitutions in different OgLuc variants.

FIG. 11 shows luminescence generated from lysed bacterial cells expressing the OgLuc variant listed in FIG. 10 using native coelenterazine ("coelenterazine"), known coelenterazine-hh ("h, h"), and novel coelenterazine PBI-3939, PBI-3945, PBI-3840, PBI-3932, PBI-3925, PBI-3894, and PBI-3896 as substrates.

FIG. 12 shows luminescence generated from lysed OgLuc variant-expressing bacterial cells using native coelenterazine ("coelenterazine"), known coelenterazine-hh ("h, h"), and novel coelenterazine PBI-3939, PBI-3945, PBI-3889, PBI-3840, PBI-3932, PBI-3925, PBI-3894, PBI-3896, and PBI-3897 as substrates.

FIG. 13 shows luminescence generated from lysed OgLuc variant-expressing bacterial cells using native coelenterazine ("coelenterazine"), known coelenterazine-hh ("H, H"), and novel coelenterazine PBI-3897, PBI-3896, and PBI-3894 as substrates.

Figure 14 shows amino acid substitutions in different OgLuc variants.

FIG. 15 shows luminescence generated from lysed bacterial cells expressing the OgLuc variant listed in FIG. 14 using native coelenterazine ("coelenterazine"), known coelenterazine-hh ("h, h"), and novel coelenterazine PBI-3897, PBI-3841, PBI-3896, and PBI-3894 as substrates.

FIG. 16 shows luminescence generated from lysed OgLuc variant-expressing bacterial cells using native coelenterazine ("coelenterazine"), known coelenterazine-H ("H"), known coelenterazine-HH ("HH"), and novel coelenterazine PBI-3841 and PBI-3897 as substrates.

FIG. 17 shows luminescence generated from lysed bacterial cells expressing the OgLuc variant and humanized Renilla (Renilla) luciferase (hRL) using native Coel ("Coel"), known Coel-hh ("h, h"), and novel Coel-sins PBI-3897 and PBI-3841 as substrates.

FIG. 18 shows luminescence generated from lysed bacterial cells expressing different OgLuc variants using native coelenterazine ("coelenterazine"), known coelenterazine-hh ("h, h"), and novel coelenterazine PBI-3939, PBI-3945, PBI-3889, and PBI-4002 as substrates.

FIG. 19 shows luminescence generated from lysed bacterial cells expressing different OgLuc variants using native coelenterazine ("coelenterazine"), known coelenterazine-H ("H, H"), known coelenterazine-hh ("H, H"), and novel coelenterazine PBI-3939, PBI-3945, PBI-3889, and PBI-4002 as substrates.

Figure 20 shows amino acid substitutions in OgLuc variants.

FIG. 21 shows luminescence generated from lysed bacterial cells expressing the OgLuc variants listed in FIG. 20 using native coelenterazine ("coelenterazine"), known coelenterazine-H ("H, H"), known coelenterazine-hh ("H, H"), and the novel coelenterazines PBI-3939, PBI-3945, PBI-4002, PBI-3932, and PBI-3840 as substrates.

Figure 22 shows amino acid substitutions in different OgLuc variants.

FIG. 23 shows luminescence generated from lysed bacterial cells expressing the OgLuc variants listed in FIG. 22 using native coelenterazine ("coelenterazine"), known coelenterazine-H ("H"), known coelenterazine-hh ("H, H"), and novel coelenterazine PBI-3939, PBI-3945, PBI-3889, PBI-4002, PBI-3932, and PBI-3840 as substrates.

FIG. 24 shows luminescence generated from lysed bacterial cells expressing different OgLuc variants and hRL ("Renilla") using native coelenterazine ("coelenterazine"), known coelenterazine-H ("H"), known coelenterazine-hh ("H, H"), and novel coelenterazine PBI-3939 and PBI-3945 as substrates.

FIG. 25 luminescence generated from lysed bacterial cells expressing different OgLuc variants using native coelenterazine ("coelenterazine"), known coelenterazine-hh ("h, h"), and novel coelenterazine PBI-3939, PBI-3945, PBI-3889, and PBI-4002 as substrates.

Figure 26 shows amino acid substitutions in different OgLuc variants.

FIG. 27 shows luminescence generated from lysed bacterial cells expressing the OgLuc variants listed in FIG. 26 using native coelenterazine ("coelenterazine"), known coelenterazine-H ("H"), known coelenterazine-hh ("H, H"), and novel coelenterazine PBI-3939, PBI-3945, PBI-3889, and PBI-4002 as substrates.

FIG. 28 shows luminescence generated from lysed bacterial cells expressing different OgLuc variants and hRL ("Renilla") using native coelenterazine ("Coel."), known coelenterazine-H ("H"), known coelenterazine-hh ("H, H"), and novel coelenterazine PBI-3939, PBI-3945, PBI-3889, and PBI-4002 as substrates.

FIG. 29 shows the luminescence of 9B8 opt and 9B8 opt + K33N in bacterial lysates using native coelenterazine and PBI-3939 as substrates, and the relative specificity of these variants for PBI-3939 compared to native coelenterazine.

FIGS. 30A-D show mutation analysis at position 166 using native coelenterazine (FIG. 30A), coelenterazine-h (FIG. 30B), and PBI-399 (FIG. 30C).

Fig. 31 shows the luminescence of the different deletions in the OgLuc variant L27V, where (-) is the machine background.

FIG. 32 shows normalized luminescence generated from lysed hRL ("Renilla") expressing HEK293 cells using native coelenterazine as a substrate, using fluorescein (BRIGHT-GLO)^TMAssay reagent) as substrate from lysed HEK293 cells expressing firefly luciferase (Luc2) and from lysed HEK293 cells expressing different OgLuc variants using the novel PBI-3939 as substrate.

FIG. 33 shows the signal stability of IV and 15C1 in bacterial lysates using novel coelenterazine PBI-3945 as the substrate and IV and 9B8 in bacterial lysates using novel coelenterazine PBI-3889 as the substrate.

Figures 34A-B show the higher activity (figure 34A) and signal stability (figure 34B) of the OgLuc variant L27V compared to firefly luciferase (Fluc) and renilla luciferase (Rluc).

FIG. 35 shows Vmax (RLU/sec) and Km (. mu.M) values for different OgLuc variants in bacterial lysates using novel coelenterazine PBI-3939 as substrate.

FIG. 36 shows Vmax (RLU/sec) and Km (. mu.M) values for different OgLuc variants in bacterial lysates using novel coelenterazine PBI-3939 as substrate.

FIG. 37 shows Vmax (RLU/sec) and Km (. mu.M) values for both 9B8 opt and 9B8 opt + K33N in bacterial lysates using novel coelenterazine PBI-3939 as the substrate.

Figure 38 shows the protein stability, luminescence at t-0 and half-life in minutes at 50 ℃ of different OgLuc variants in bacterial lysates using native coelenterazine as substrate.

Figures 39A-B show the structural integrity (as determined by expression, stability and solubility, as shown by SDS-PAGE analysis) of different OgLuc variants in bacterial lysates at 25 ℃ (figure 39A) and 37 ℃ (figure 39B) compared to renilla luciferase (hRL) and firefly luciferase (Luc 2).

FIGS. 40A-B show the protein stability at 60 ℃ of 9B8 opt and 9B8 opt + K33N in bacterial lysates using novel coelenterazine PBI-3939 as the substrate, the natural logarithm (ln) of luminescence (in RLU) over time (FIG. 40A) and the half-life in hours (FIG. 40B).

Figure 41 shows the percentage of activity of OgLuc variants 9B8 and L27V at 60 ℃.

Fig. 42A-B show the protein stability of OgLuc variant L27V at different pH (fig. 42A) and salt concentrations (fig. 42B).

FIGS. 43A-B show gel filtered spectral analysis of purified C1+ A4E (FIG. 43A) and 9B8 (FIG. 43B).

Figure 44 shows gel filtration chromatography analysis indicating that OgLuc variant L27V exists as a monomer.

FIGS. 45A-B show the analysis by SDS-PAGE (FIG. 45A) of undiluted and 1:1 diluted bacterial lysate samples Different OgLuc variants-

(HT7) protein expression level of fusion protein, and normalized protein expression level (fig. 45B).

Fig. 46A-B show protein expression (fig. 46A) and solubility (fig. 46B) of OgLuc variants 9B8 opt, V2, and L27V.

FIG. 47 shows the normalized luminescence in RLU generated from lysed HEK293 cells expressing IV, 9B8 and hRL ("Renilla") using native coelenterazine and novel coelenterazine PBI-3939 as substrates.

FIG. 48 shows the utilization of PBI-3939, Luciferin (BRIGHT-GLO), respectively^TMAssay reagents) and native coelenterazine as substrates from lysed HEK293 cells expressing pF4Ag-Ogluc-9B8-HT7, pF4Ag-Luc2-HT7 and pF4Ag-Renilla-HT 7.

FIG. 49 shows the luminescence generated from lysed HEK293 cells expressing 30ng or 100ng of plasmid DNA encoding 9B8 opt or 9B8 opt + K33N ("K33N") using novel coelenterazine PBI-3939 as a substrate.

FIGS. 50A-E show luminescence of the OgLuc variant L27V in HEK293 cells (FIG. 50A) and HeLa cells (unfused) (FIG. 50B) compared to firefly luciferase (Luc2), and in HEK293 ("HEK") and HeLa cells ("HeLa") compared to the OgLuc variant L27V (FIG. 50C) and firefly luciferase (Luc2) (FIG. 50D)

Luminescence of the fusion, and

firefly luciferase (Luc2) comparison

-protein expression of OgLuc L27V.

Fig. 51 shows inhibition assays against the LOPAC library OgLuc variants 9B8 and L27V to determine their sensitivity to off-target interactions.

Fig. 52A-E show the inhibition analysis of OgLuc variants 9B8 and L27V by suramin and tyrag 835 (fig. 52A-C) and the chemical structures of suramin (fig. 52D) and tyrag 835 (fig. 52E).

Figure 53 shows analysis of the activity of OgLuc variants 9B8 and L27V in the presence of BSA to determine resistance to non-specific protein interactions.

Figure 54 shows the percent activity of OgLuc variants 9B8 and L27V to determine reactivity to plastics.

Figure 55 shows the luminescence generated from lysed HEK293 cells expressing the IV cAMP transcription reporter when treated with forskolin ("induced") and not treated with forskolin ("basal") and the fold induction due to forskolin ("fold") compared to hRL ("renilla") using known coelenterazine-h as substrate.

FIG. 56 shows the use of PBI-3939 (for 9B8 and 9B8 opt), native coelenterazine (for hRL), or fluorescein (BRIGHT-GLO)^TMA detection reagent; for Luc2) as substrate from lysed HEK293 cells expressing 9B8, 9B8 opt, hRL ("renilla") or firefly luciferase ("Luc 2") cAMP transcription reporters, and fold induction (response) ("fold") due to forskolin treatment.

FIG. 57 shows the luminescence generated from lysed HEK293 cells expressing 9B8 opt and 9B8 opt + K33N ("K33N") cAMP transcription reporters, and fold induction due to forskolin treatment ("fold induction"), using novel coelenterazine PBI-3939 as a substrate, with ("induced") or without forskolin treatment ("basal").

FIGS. 58A-C show luminescence of OgLuc variant 9B8 and L27V lytic reporter constructs for multiple pathways in multiple cell types.

FIGS. 59A-C show the luminescence of the OgLuc variant L27V reporter construct in different cell lines and with different response elements.

FIGS. 60A-B show luminescence of the OgLuc variant L27V secretable reporter in comparison to Metridia longa luciferase with a CMV promoter (FIG. 60A) or NFkB response element (FIG. 60B).

Fig. 61A-F show absolute luminescence (fig. 61A and 61B), normalized luminescence (fig. 61C and 61D), and fold response (fig. 61E and 61F) of the optimized form of L27V (L27V01, L27V02, and L27V03) compared to L27V (L27V00) expressed in HeLa cells.

Fig. 62A-B show the luminescence of secreted OgLuc variant L27V02 (containing IL-6 secretion signals) reporter (fig. 62A) and L27V02 ("L27V (02)"), L27V02P ("L27V (02) P (01)") and luc2 ("Fluc") reporter expressed in HepG2 cells treated with different doses of rhTNF α ("TNF α").

Figure 63 shows the luminescence generated from culture medium and lysate samples of HEK293 cells expressing codon optimized variant IV opt with or without secretion signal sequence using the novel PBI-3939 as substrate compared to hRL ("renilla") with or without secretion signal sequence using native coelenterazine as substrate.

Fig. 64A-D show the luminescence of secreted OgLuc variants 9B8, V2, and L27V reporter expressed in CHO cells (fig. 64A and 64B) and HeLa cells (fig. 64C and 64D).

FIGS. 65A-B show the luminescence and use of Ready-to-Glow generated from secreted OgLuc variants 9B8 and V2 using PBI-3939 as a substrate^TMComparison of luminescence of secreted luciferase from daphnia elongata as substrate numerically (FIG. 65A) and graphically (FIG. 65B).

FIGS. 66A-B show the use of the coelenterazine derivative ENDUREN^TM(FIG. 66A) and VIVIVIN^TM(FIG. 66B) and novel coelenterazine PBI-3939 (FIG. 66B) as substrates increased fold of luminescence above background generated from HEK293 cells expressing hRL ("Ren") and 9B8 opt.

FIGS. 67A-D show transient expression of L27V-

U2OS cells of fusions (FIG. 67A) or IL6-L27V fusions (FIGS. 67B-D)Confocal images of (a). Scale bar 20 μm.

Figure 68 shows the luminescence generated from lysed bacterial cells expressing different OgLuc variants and hRL ("renilla") in the presence ("sandd") or absence (pF4Ag) of the sandwich background using native coelenterazine as substrate.

Figure 69 shows the fold reduction in activity of different OgLuc variants and hRL ("renilla") due to the presence of a sandwich background using native coelenterazine as a substrate.

Figure 70 shows that the presence of sandwich background using the novel coelenterazine PBI-3939 as substrate results in a fold reduction in the activity of 9B8 opt and 9B8 opt + K33N in bacterial lysates.

Fig. 71 shows a spectral profile of the OgLuc variant L27V.

Fig. 72 shows the luminescence of the two cycle-exchanged (CP) forms of the OgLuc variants L27V, CP84, and CP95 without a linker or a linker with 5, 10, or 20 amino acids.

FIGS. 73A-G show the luminescence of different CP-TEV protease L27V constructs expressed in wheat germ extracts (FIGS. 73A-D), E.coli (FIGS. 73F-G), and HEK 293 cells (FIG. 73H). FIGS. 73A-D show the substrate luminescence of different CP-TEV protease L27V constructs prior to TEV addition. FIG. 73E shows the response of the CP-TEV protease L27V construct of FIGS. 73A-D.

Figure 74 shows the fold response of different protein complementary L27V pairs.

FIGS. 75A-C show the luminescence of a different pair of Protein Complementation (PCA) L27V, which is the pair of protein complementation L27V: one L27V fragment of each pair was fused to FKBP or FRB using either the 1/4 configuration (FIG. 75A) or the 2/3 configuration (FIG. 75B), and the interaction of FKBP and FRB monitored in HEK293 cells. The luminescence of the different Protein Complementation (PCA) negative controls was also monitored (fig. 75C).

FIGS. 76A-H show the luminescence of a different pair of Protein Complementation (PCA) L27V, which is the pair of protein complementation L27V: one L27V fragment of each pair was fused to FKBP or FRB using either the 2/3 configuration (FIGS. 76A and 76C) or the 1/4 configuration (FIGS. 76B and 76D), and the interactions of FKBP and FRB monitored in wheat germ extracts (FIGS. 76A and 76B) and Rabbit Reticulocyte Lysates (RRL) (FIGS. 76C and 76D). Luminescence of different Protein Complementation (PCA) negative controls was also measured in the cell-free system (fig. 76E). The 1/4 configuration was used in cell-free systems (fig. 76F), HEK293 cells (fig. 76G) and lysis systems (fig. 76H).

FIGS. 77A-C show luminescence from different pairs of protein-complementary L27V treated with FK506 and rapamycin (FIG. 77A), and the chemical structures of FK506 (FIG. 77A) and rapamycin (FIG. 77B).

Figure 78 shows the activity of treatment of OgLuc variant 9B8 cAMP bioreceptors with forskolin.

FIGS. 79A-D show luminescence of circularly permuted (FIGS. 79A and 79C) and directly dividing (FIGS. 79B and 79D) L27V variants in rabbit reticulocyte lysates (FIGS. 79A-B) and HEK293 cells (FIGS. 79C-D).

Fig. 80A-B show the subcellular distribution of the OgLuc variant L27V (fig. 80A) and the control vector pGEM3ZF (fig. 80B) in U2OS cells for different exposure times.

Figures 81A-C show subcellular localization of OgLuc variant L27V fused to transcription factor Nrf2 (figure 81B) or GPCR (figure 81C) compared to unfused L27V control (figure 81A).

FIGS. 82A-C show the use of PBI-4377 OgLuc variant 9B8 opt to monitor intracellular signaling pathways (FIG. 82A). 9B8 opt luciferase was fused to IkB (FIG. 82B) or ODD (oxygen dependent degradation domain of Hif-1. alpha.) (FIG. 82C) and fold response to stimuli (TNF. alpha. for IkB and phenanthroline for ODD) was monitored by luminescence.

Fig. 83A-C show monitoring of oxidative stress signaling pathways using OgLuc variants (fig. 83A), L27V02 (fig. 83B), or renilla luciferase (Rluc) (fig. 83C).

FIGS. 84A-B show a comparison of Nrf2-L27V02 receptors (FIG. 84A) and the Nrf2(ARE) -Luc2P reporter (FIG. 84B).

FIGS. 85A-B show the emission spectra of IV-HT7 with and without ligand using 1. mu.M TMR (FIG. 85A) or 10. mu.M rhodamine 110 (FIG. 85B) as the ligand for HT7 and coelenterazine-h as the substrate for IV.

FIG. 86 shows luminescence generated from lysed 9B8 opt-expressing bacterial cells mixed with caspase-3 ("+ caspase") or not mixed with caspase-3 ("caspase-free") using the precore enteron substrate.

FIGS. 87A-C show the luminescence generated from circularly permuted, directly cleaved L27V variants CP84 and CP103, treated with rapamycin (FIG. 87B) or without rapamycin (not shown), using PBI-3939 as a substrate, and the response due to rapamycin treatment (FIG. 87C). The concept of a directly split variant of cycle swapping is shown in fig. 87A.

Figure 88 shows the percent remaining activity of the L27V variant after exposure to different amounts of urea.

Figure 89 shows the effect of 3M urea on the activity of the L27V variant.

FIGS. 90A-B show bioluminescence imaging of Nuclear Receptor (NR) translocation of the OgLuc fusion induced by PBI-3939 substrate hormone.

FIGS. 91A-B show bioluminescent imaging of protein kinase C α (PKC α) translocation of OgLuc fusion induced by phorbol ester substrate PBI-3939.

FIGS. 92A-B show bioluminescence imaging of autophagosome protein translocation using PBI-3939 substrate OgLuc fusion.

Detailed description of the invention

Before any embodiments of the invention are explained in detail, it is to be understood that the invention is not limited in its application to the details of construction, synthesis, and arrangement of components set forth in the following description or illustrated in the following drawings. The invention has been described with respect to particular embodiments and techniques, however, the invention is capable of other embodiments and of being practiced or of being carried out in various ways.

In the following description of the process of the invention, the process steps are carried out at room temperature (about 22 ℃) and atmospheric pressure, unless otherwise stated. It should also be expressly understood that any numerical range recited herein includes all values from the lower value to the upper value. For example, if a concentration range or beneficial effect range is indicated as 1% to 50%, it is intended that such ranges as 2% to 40%, 10% to 30%, or 1% to 3%, etc. are expressly enumerated in this specification. Similarly, if a given sequence identity range is, for example, between 60% and < 100%, it is intended that such ranges as 65%, 75%, 85%, 90%, 95%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible values from the lowest value to the highest value are to be considered to be expressly stated in this specification.

Unless expressly stated otherwise, the term "comprising" in the context of this application is intended to indicate that there may optionally be additional members other than the members of the list introduced by "comprising". However, it is contemplated that the term "comprising" encompasses the possibility that no additional member is present as a particular embodiment of the present invention, i.e., that embodiment is intended to be understood as having the meaning of "consisting of … …".

The following detailed description discloses specific and/or preferred variations of individual features of the invention. The present invention also contemplates as particularly preferred embodiments those embodiments produced by combining two or more of the specific and/or preferred variations described with respect to two or more of the features of the present invention.

All indications of relative amounts in this application are made on a weight/weight basis unless expressly stated otherwise. The relative amounts of the components characterized by general terms are intended to refer to all specific variations or the total number of members encompassed by the general term. If it is stated that a certain component defined by a general term is present in a certain relative amount, and if the component is also characterized as a specific variation or member encompassed by that general term, it is intended that no further variation or member encompassed by that general term is otherwise present, such that the total relative amount of the component encompassed by that general term exceeds the specified relative amount. More preferably, additional variations or members encompassed by the generic term are not present at all.

Overview

In various aspects, the invention relates to novel compounds, novel luciferases and combinations thereof. The present invention encompasses methods, compositions and kits comprising the novel compounds, novel luciferases and/or combinations thereof.

The novel compounds are novel coelenterazines that can be used as substrates for the utilization of coelenterazines to produce luminescent proteins including, but not limited to, luciferases and luminescent proteins found in different marine organisms such as echinocyte luciferase (e.g., renilla luciferases), jellyfish luciferase (e.g., jellyfish luminescent protein from Aequorea jellyfish) luciferase and decapod luciferase (e.g., luciferase complex of aenodus gracilis). In various embodiments, the novel coelenterazine of the present invention has at least one of enhanced physical stability (e.g., enhanced coelenterazine stability), reduced autoluminescence, and increased biocompatibility with cells (e.g., less toxicity to cells, including heterologous cell types) relative to native coelenterazine.

The novel luciferases disclosed herein include variants of the active subunit of decapod luciferases. The novel luciferases can utilize different coelenterazines as substrates, including, but not limited to, native and known coelenterazines as well as the novel coelenterazines of the present invention. The novel luciferase exhibits at least one of the following: enhanced luminescence (including increased brightness, enhanced signal stability, and/or signal duration); enhanced enzyme stability (i.e., enhanced enzyme activity, including enhanced tolerance to elevated temperature, changes in pH, inhibitors, denaturants, and/or detergents); altered substrate specificity (i.e., a change in relative substrate specificity); and enhanced biocompatibility (including at least one of increased expression in cells, reduced toxicity, and/or cellular stress). In various embodiments, the invention encompasses novel luciferases that exist in solution as soluble active monomers or chemically linked to other molecules (e.g., fusion proteins), or attached to solid surfaces (e.g., particles, capillaries, or detection tubes or plates).

Certain combinations of novel coelenterazine and novel luciferases provide significant technical advantages for bioluminescent assays, including enhanced luminescence, wherein the enhanced luminescence may be due to one or more factors including enhanced signal stability and enhanced stability of coelenterazine. In addition, many of the novel coelenterazines are designed to be smaller than commercially available and/or known coelenterazines. In certain instances, the novel luciferases of the invention preferably utilize novel, smaller coelenterazines over commercially available and/or known larger coelenterazines.

The present invention encompasses the following combinations: novel luciferase variants with novel coelenterazine; novel luciferase variants with known or native coelenterazine; and novel coelenterazine and any known or native protein (e.g., luciferase or photoprotein) that uses coelenterazine as a substrate.

The term "coelenterazine" refers to naturally occurring ("natural") coelenterazine and analogs thereof, including coelenterazine-n, coelenterazine-f, coelenterazine-h, coelenterazine-hcp, coelenterazine-cp, coelenterazine-c, coelenterazine-e, coelenterazine-fcp, dideoxy coelenterazine ("coelenterazine-hh"), coelenterazine-i, coelenterazine-icp, coelenterazine-v, and 2-methyl coelenterazine, as well as those disclosed in WO 2003/040100 and U.S. application Ser. No. 12/056,073 (paragraph [0086], the disclosures of which are incorporated herein by reference. The term "coelenterazine" also refers to the novel coelenterazine disclosed herein (see below). The term "known coelenterazine" refers to analogs of coelenterazine known prior to the present invention.

The term "OgLuc" refers to the decapod luciferase protein, or a variant of this protein, that generates light in the presence of coelenterazine. The OgLuc protein may be a monomer in its naturally occurring form or may be a subunit of a protein complex. OgLuc used in the exemplary embodiments disclosed herein is the 19kDa subunit of the luciferase complex from Acrophorus gracilis, but similar polypeptides from other decapod species (including other Acrophorus species) may also be used and encompassed by the present invention (see R.D. Dennell, Observations on the luminescence of the nutritional Crustacea of the Bermuda area, Zool.J. Linn. Soc., Lond.42(1955), pages 393-406; see also Poupin et al, 9.1999. Inventiai documente des et bilan des formes les plus communes de la mer d'Iroise.Rapport Scientifique du Laboratoire d'Océanographie de

Navale (LOEN), Brest (page 83), each of which is incorporated herein by reference); examples include, but are not limited to, the following luciferases: the family of the aenopsis (Aristeidae) including penaeus brachypus (Plesiopenaeus ruduscans); pandalidea family, including Allophyllus spp (Heterocarpus spp.) and Parapanadalus richardi, Strongyloideae (Solenoceridae), including Hymenopenaeus bideis and Tropical metapenaeus chinensis; the family of the fireflies (Luciferidae), including positive-type fireflies (Lucifer typus); sergestidae (Sergestidae) including Charophytus maxima (Sergestes atlanticus), Scophytus arctica (Sergestes arcticus), Cyanea acuta (Sergestes armatus), Sergestes pedformis (Sergestes armenius), Sergestes cornutus, Sergestes edwards, Sergestes hensenensis, Cydonia pectinifera (Sergestes pectus), Sergestes sargi, Cydonia formosanus (Sergestes sementis), Sergestis sempervirgula, Sergestissa charliensis, Sergestis grandis, Sergestis cerasus (Sergestis lucens), Sergestis ruber (Sergestis), Sergestis punctilex, Sergestissa robusta, Sergestis scintillation cerasus (Sergestis), and Sergestella; the family of glassshrimp (Pasiphaeidae) including Glyphus marsuphialis, Paecilomyces argenteus (Leptochlea bermudensis), Gouqua glaucus (Parapasiphae sulcations), and Pasiphea tarda; oplophoraceae (Oplophoridae) including Acanthophyta acanthis, Acanthophyta acutiferons, Acanthophyta breviaristis, Acanthophyta cucula, short angled acutus (Acanthophyta curustris), abnormal acutus (Acanthophyta eximia), Acanthophyta gracilides, Acanthophyta kinensis, Acanthophyta purpurea, Acanthophyta pallea, Acanthophyta media, Acanthophyta microphylla, Acantha vitrulata, Acantha pelagica, Acanthophyta Prionita, Acanthus purpurea, Acanthus erythrophyllus praerus, Acanthus erythrosticus erythropolis, Acanthus erythrosticus sanguinea, Acanthus autophycus, Acanthus gigaea, Acanthus erythroptera, Ephyhythora purpurea, Ephybrida membranes (Ephybrida membranes), Ephybrida membranes, and Achythora hybrida membranes iles), Meningodora micella, soft pockmarked shrimps (Meningodora mollis), Meningodora vesca, carina carinata (Nototomus gibbosus), Nototomus auriculus, Ceratopterus gracilis, Oplophorus grimalindii, Oplophorus novaezeaanallae, Oplophorus spicata, Oplophorus folicaeus, Oplophorus spissus, Spathous punctatus (Oplophorus typus), Systella brauris, Systella crista, Systella clavis dementis (Systella dendrons) and Spathophysa perus giganteus (Systella pellucida); and the family Penaeidae (Thalassociaceae), including Strongylocentrotus spinosus (Chlorotocoids spinouda), Stenopsis chinensis (Thalassocias crinita) and Thalassocias lucida.

The polypeptide sequence of the 19kDa subunit of the mature (i.e.no signal sequence) Nitraria gracilis luciferase (i.e.169 amino acids from residue 28 to residue 196 of BAB 13776) is given in SEQ ID NO 1. In various embodiments, methionine and valine residues are inserted at the beginning of the synthetic OgLuc sequence (e.g., if shown in the C1+ A4E polypeptide sequence SEQ ID NO: 3) to facilitate cloning and expression in a heterologous system. However, for consistency, the position numbering of the various amino acid substitutions referred to herein is "relative to" SEQ ID NO:1, i.e., the polypeptide sequence of the native 19kDa subunit of the mature (NO signal sequence), Nitraria gracilis luciferase protein complex.

In particular, a decapod luciferase if the protein has a sequence identity of > 30%, preferably > 40%, and most preferably > 50% based on an alignment of its amino acid sequence to SEQ ID No. 1, and the protein can use coelenterazine as a substrate to catalyze the emission of luminescence, and the amino acid sequence starting at the position corresponding to position 8 of SEQ ID No. 1 is:

[GSAIVK]-{FE}-[FYW]-x-[LIVMFSYQ]-x-x-{K}-x-[NHGK]-x-[ DE]-x-[LIVMFY]-[LIVMWF]-x-{G}-[LIVMAKRG](SEQ ID NO. 330)(VII)，

with no more than 5 differences, or more preferably no more than 4, 3, 2 or 1 difference, or most preferably no difference, wherein the differences occur in positions corresponding to mode positions 1, 2, 3, 5, 8, 10, 12, 14, 15, 17 or 18 of formula (VII) according to table 4. The differences may also include gaps or insertions between the mode positions of table 4.

The term "variant" refers to a modified form of the starting polypeptide or polynucleotide sequence. The term "parent" is a relative term and refers to the starting sequence as modified thereby. The parent sequence is generally used as a reference for the protein encoded by the resulting modified sequence, e.g., to compare the activity levels or other properties of the proteins encoded by the parent and modified sequences. The starting sequence may be a naturally occurring (i.e., native or wild-type) sequence. The starting sequence may also be a variant sequence which is thus further modified. A polypeptide sequence is "modified" when one or more amino acids (which may be naturally occurring or synthetic) are substituted, deleted and/or added at the beginning, middle and/or end of the sequence. A polynucleotide sequence is "modified" when one or more nucleotides are substituted, deleted, and/or added at the beginning, middle, and/or end of the sequence, but the amino acids encoded by the sequence may or may not be changed. In some embodiments, the modification results in the variant being a functional fragment of a particular OgLuc or OgLuc variant. A functional fragment is a fragment that has the same functional activity as the full parent sequence but less than the full parent sequence. Functional activity is the ability to exhibit luminescence. In some embodiments, the modification results in a variant of the exchange sequence that is a parent sequence, such as a circular exchange sequence and an exchange sequence that includes deletions and/or insertions.

Some of the OgLuc variants disclosed herein have been assigned abbreviated names to aid in discussion. The term "C1 + A4E" (also referred to as "C1 A4E") refers to OgLuc variants (SEQ ID NOs: 2 and 3) having the amino acid substitutions A4E, Q11R, a33K, V44I, a54F, P115E, Q124K, Y138I, and N166R relative to SEQ ID NO:1 (wherein the format "x # Y" refers to the parent amino acid "x" substituted as the "#" position of the variant amino acid "Y"). Unless otherwise indicated, variants of the C1+ A4E OgLuc variants presented herein comprise at least the amino acid substitutions found in C1+ A4E. The term "IVY" refers to variants of the C1+ A4E OgLuc variant (SEQ ID NOS: 8 and 9) having additional amino acid substitutions F54I, I90V and F77Y relative to SEQ ID NO: 1. The term "IV" refers to another variant of the C1+ A4E OgLuc variant (SEQ ID NOS: 14 and 15) having additional amino acid substitutions F54I and I90V relative to SEQ ID NO: 1. The term "QC 27" refers to yet another variant of the C1+ A4E OgLuc variant (SEQ ID NOs: 4 and 5) having additional amino acid substitutions Q18L, F54I, L92H and Y109F relative to SEQ ID NO: 1. The term "QC 27-9 a" refers to variants of the QC27 OgLuc variant (SEQ ID NOS: 6 and 7) with additional amino acid substitutions V21L, F68Y, L72Q, M75K, H92R and V158F relative to SEQ ID NO: 1. The term "9B 8" refers to variants of the IV OgLuc variant (SEQ ID NOS: 18 and 19) having additional amino acid substitutions Q18L, F68Y, L72Q and M75K relative to SEQ ID NO: 1. The term "9B 8 opt" refers to a codon optimized form of the 9B8 variant (SEQ ID NO: 24). The term "9B 8 opt + K33N" refers to variants of the 9B8 opt variant (SEQ ID NOS: 42 and 43) having additional amino acid substitutions K33N relative to SEQ ID NO: 1. The term "9B 8 opt + K33N + 170G" refers to variants having an additional glycine appended to the C-terminus of the variant relative to SEQ ID NO:1, i.e., the "9B 8 opt + K33N" variant of 170G (SEQ ID NO:68 and 69). The terms "L27V + T39T + K43R + Y68D" and "L27V" refer to variants of the "9B 8 opt + K33N" variant (SEQ ID NOS: 88 and 89) having additional amino acid substitutions L27V, T39T, K43R and Y68D relative to SEQ ID NO: 1. The terms "T39T + K43R + Y68D" and "V2" refer to variants of the "9B 8 opt + K33N" variant (SEQ ID NOS: 92 and 93) having additional amino acid substitutions T39T, K43R and Y68D relative to SEQ ID NO: 1.

By "enhanced" is meant an increase in a particular property (e.g., luminescence, signal stability, biocompatibility, protein stability (e.g., enzyme stability), or protein expression) relative to a reference luciferase plus coelenterazine combination or the luciferase in question, wherein the increase is at least 1%, at least 5%, at least 10%, at least 20%, at least 25%, at least 50%, at least 75%, at least 90%, at least 100%, at least 200%, at least 500%, or at least 1000% above the reference luciferase plus coelenterazine combination or the luciferase in question.

The term "luminescence" refers to the light output of the OgLuc variant under appropriate conditions, e.g., in the presence of an appropriate substrate such as coelenterazine. The light output can be measured as an instantaneous or near instantaneous measure of light output at the start of the luminescence reaction (sometimes referred to as "T ═ 0" luminescence or "flash"), which can begin after addition of the coelenterazine substrate. In various embodiments, the luminescent reaction is carried out in solution. In a further embodiment, the luminescent reaction is carried out on a solid support. The solution may comprise a lysate, for example from cells in a prokaryotic or eukaryotic expression system. In other embodiments, expression occurs in a cell-free system, or the luciferase protein is secreted into the extracellular medium, so that, in the latter case, it is not necessary to produce a lysate. In some embodiments, the reaction is initiated by injecting an appropriate starting material, e.g., coelenterazine, a buffer, etc., into a reaction chamber (e.g., a well of a multi-well plate, such as a 96-well plate) containing the photoprotein. In still further embodiments, the OgLuc variant and/or novel coelenterazine is introduced into a host and a measurement of luminescence is performed on the host or a portion thereof, which may include the whole organism or a cell, tissue, explant, or extract thereof. The reaction chamber may be located in a reading device that can measure the light output, for example, with a photometer or photomultiplier. The light output or luminescence can also be measured over time, e.g. in the same reaction chamber for a period of seconds, minutes, hours, etc. The light output or emission may be recorded as an average over time, half-life of decay of the signal, sum of the signals over time, or maximum output. Luminescence can be measured in Relative Light Units (RLU).

The "enhanced luminescence" of the OgLuc variant may be due to one or more of the following characteristics: enhanced light output (i.e., brightness), enhanced substrate specificity, enhanced signal stability, and/or enhanced signal duration. Enhanced signal stability includes an increase in the time for which the signal from the luciferase continues to emit light, e.g., as measured by the half-life of the decay of the signal over time. Enhanced luminescence can be relative to luciferases such as wild-type OgLuc, OgLuc variant proteins, renilla luciferases (e.g., hRluc) or firefly luciferases (e.g., from h. luc) combined with native, known or novel substratesComparable properties of Luc2 luciferase from North American firefly (Photinus pyralis), as shown in the examples below. For example, the luminescence of a given OgLuc variant in combination with a particular coelenterazine (including native, known, or novel coelenterazine) can be compared to the properties of one of the OgLuc variants C1+ A4E, IV, or IVY in combination with any of the native, known, or novel coelenterazines disclosed herein, using one or more of the assays disclosed in the examples below. In particular, the enhanced luminescence can be determined by measuring the luminescence signal (RLU) generated by incubation of the bacterial lysate containing the OgLuc variant in question with the substrate PBI-3939. Measurements are taken at locations that may include TERGITOL ^TMTo provide Glo-like kinetics, e.g., in which enzyme inactivation is slowed and luminescent signals are stabilized, which are described elsewhere in this application. In some embodiments, some luciferase variants, e.g., L27V, with certain compounds, e.g., PBI-3939, provide for extended duration of luminescent emission, or in the absence of TERGITOL^TMGlow-like kinetics occur. The luminescent signal can be compared to the luminescent signal of a reference point such as the C1+ A4E variant to coelenterazine or coelenterazine-h or renilla luciferase to native coelenterazine.

"enzyme stability" refers to the stability of an enzyme activity (i.e., the tolerance of the enzyme activity to reaction conditions). Enhanced enzyme stability refers to enhanced stability of the enzyme activity (i.e., enhanced tolerance to reaction conditions). Enhanced enzyme stability includes enhanced thermostability (e.g., stability at elevated temperatures) and chemical stability (e.g., in inhibitors or denaturants such as detergents (including, e.g., TRITON)^TMX-100) in the presence). Enzyme stability can be used as a measure of protein stability, particularly under conditions known to disrupt protein structure, such as high temperature or in the presence of chemical denaturants. In particular, enhanced protein stability can be determined using thermal analysis as described elsewhere in this application (e.g., in example 28). The luminescent signal can be compared to a reference point for the C1+ A4E variant and coelenterazine or coelenterazine-h or renilla luciferase and native coelenterazine.

"biocompatibility" refers to the tolerance of a cell (e.g., prokaryotic or eukaryotic) to luciferase and/or coelenterazine compounds. The biocompatibility of luciferase and/or coelenterazine is related to the stress it causes on the host cells. For example, a luciferase that is not tolerated by the cell may not be efficiently expressed in the cell, e.g., the luciferase may be expressed in the cell but exhibits reduced activity due to the formation of inclusion bodies of the expressed protein. The biocompatibility of luciferase is associated with the ability of the cell to tolerate the insertion of an exogenous gene, i.e., a transgene comprising a gene encoding luciferase or a fragment thereof, whereby the cell with the transgene does not exhibit manifestations of stress including induction of stress response pathways, reduced growth rate, and/or reduced viability (e.g., reduced number of viable cells, reduced membrane integrity, or increased rate of apoptosis). Other indications of cellular stress may include alterations in gene expression, signaling pathways, and/or regulatory pathways. The enhanced biocompatibility of the OgLuc variants can result from factors such as enhanced protein expression and/or reduced cellular stress. Expression for enhanced luminescence of a particular polynucleotide encoding an OgLuc variant can be determined relative to the luminescence expression level of a polynucleotide encoding a wild-type OgLuc or an OgLuc variant protein (including codon-optimized polynucleotides), wherein luminescence activity can be used as a means to monitor protein expression levels.

In particular, the enhanced biocompatibility of the OgLuc variants, novel coelenterazine compounds, and/or combinations thereof can be determined by measuring cell viability and/or growth rate of the cells. For example, enhanced biocompatibility of an OgLuc variant can be determined by measuring the viability and/or growth rate of cells comprising the OgLuc variant as compared to cells comprising a firefly luciferase or a renilla luciferase, or no luciferase, in the absence of any coelenterazine compound to determine the degree of compatibility and/or toxicity of the luciferase to the cells. The enhanced biocompatibility of the novel coelenterazine compounds may be determined by measuring the viability of cells exposed to the novel coelenterazine compounds as compared to native or known coelenterazine in the absence of luciferase expression by the cells to determine the degree of compatibility and/or toxicity of the coelenterazine compounds to the cells. The enhanced biocompatibility of the combination of the OgLuc variant and the novel coelenterazine compound can be determined by measuring cell viability and/or growth rate of the cells comprising the OgLuc variant and exposed to the novel coelenterazine as compared to cells comprising firefly luciferase or renilla luciferase or no luciferase and exposed to native or known coelenterazine.

In particular, enhanced biocompatibility may utilize cell viability assays as described elsewhere in this application (e.g., utilizing CELLTITER-

Determination or utilization of CASPASE-

Technical apoptosis assays) or assays known in the art. The effect of the OgLuc variant on cell viability or apoptosis can be compared to the effect of a reference luciferase such as the C1+ A4E variant, a firefly luciferase, or a renilla luciferase. The effect of the novel coelenterazine compounds on cell viability or apoptosis may be compared to the effect of natural or known coelenterazine compounds on cell viability or apoptosis.

Enhanced biocompatibility can be determined by measuring the effect of OgLuc variants and/or novel coelenterazine compounds on cell growth or gene expression. For example, the enhanced biocompatibility of the OgLuc variant can be determined by measuring the number of cells after a period of time or by determining the expression of a stress response gene in a sample of cells comprising the OgLuc variant as compared to cells comprising another luciferase or without luciferase. The enhanced biocompatibility of the novel coelenterazine compounds may be determined by measuring the number of cells after a period of time or by measuring the expression of stress response genes in a sample of cells exposed to the novel coelenterazine compounds as compared to cells exposed to native or known coelenterazine or not. The effect of the OgLuc variant on cell growth or gene expression can be compared to a reference luciferase, such as the C1+ A4E variant, a firefly luciferase, or a renilla luciferase. The effect of the novel coelenterazine on cell growth or gene expression may be compared to native or known coelenterazines.

The bright signal of luciferase and the small size of the OgLuc gene can facilitate the identification of robust, stable cell lines expressing the cytoplasmic or secreted forms of the OgLuc variants of the invention. It is expected that a relatively small gene sequence will reduce the likelihood of gene instability resulting from integration of exogenous DNA into the genome of a cell. The enhanced biocompatibility of the OgLuc variants and/or novel coelenterazine is facilitated by the increased brightness, less protein expression, and thus less DNA required for transfection of the present OgLuc variants and/or novel coelenterazine, which may result in a given level of brightness, as compared to other known luciferases such as native OgLuc, firefly luciferase, or renilla luciferase. The enhanced biocompatibility of the OgLuc variants can be measured by the amount of DNA or reagents required in the transient transfection to produce cells with the same level of luminescence as cells transfected with other luciferases, such as native OgLuc, firefly luciferase or renilla luciferase, for example transfection chemicals. In some embodiments, the amount of OgLuc variant DNA or reagent required for transfection to generate transfected cells with the same level of luminescence obtained with other luciferases is less than that required for another luciferase, e.g., a native OgLuc, firefly luciferase, or renilla luciferase. The enhanced biocompatibility of the OgLuc variant can be measured by the recovery time of the cells after transfection. In some embodiments, the amount of time required for recovery after transfection with the OgLuc variant is less than the time required for another luciferase, such as a native OgLuc, firefly luciferase, or renilla luciferase.

"relative substrate specificity" is determined by measuring the luminescence of the luciferase in the presence of coelenterazine substrate divided by the luminescence of the luciferase in the presence of reference coelenterazine. For example, relative specificity may be determined by dividing the luminescence of a luciferase utilising a novel coelenterazine of the invention by the luminescence of a luciferase utilising a different coelenterazine (e.g. a native or known coelenterazine, see figure 1 for example, or a different novel coelenterazine of the invention). The test coelenterazine substrate and the compared reference coelenterazine substrate are considered as a comparison substrate pair for determining the relative substrate specificity.

The "change in relative substrate specificity" is determined by dividing the relative substrate specificity of a test luciferase using a comparison substrate pair by the relative substrate specificity of a reference luciferase using the same comparison substrate pair. For example, the change in relative specificity can be determined by dividing the relative substrate specificity of a test luciferase using the novel coelenterazine of the invention as compared to a different coelenterazine (e.g., a native or known coelenterazine or a different novel coelenterazine of the invention) by the relative substrate specificity of a reference luciferase using the novel coelenterazine of the invention as compared to the same different coelenterazine used to test the luciferase.

In some embodiments, the luminescence with one novel coelenterazine is compared to the luminescence with a different novel coelenterazine. In some embodiments, the luminescence using one native or known coelenterazine is compared to the luminescence using another native or known coelenterazine. In still further embodiments, the luminescence using a native or known coelenterazine is compared to the luminescence using the novel coelenterazine.

The novel coelenterazine of the present invention includes properties such as enhanced physical stability (e.g., enhanced coelenterazine stability) or reduced self luminescence. Physical stability of coelenterazine refers to the ability of coelenterazine to be stable under conditions such that it maintains luminescence when used as a substrate for luciferase. Luminescence independent of the activity of luciferase or photoprotein is called self luminescence. Spontaneous light emission is the emission of a substance by the energy released in the form of light during decay or decomposition. For example, self-luminescence may result from the spontaneous oxidation of the substrate coelenterazine that activates luminescence.

As used herein, "pure" or "purified" means that the subject substance is the predominant substance present (i.e., on a molar and/or mass basis, more abundant than any other individual substance, except for water, solvents, buffers, or other common components of the aqueous system in the composition), and, in some embodiments, the purified fraction is a composition in which the subject substance comprises at least about 50% (on a molar basis) of all macromolecular substances present. Generally, a "substantially pure" composition will comprise greater than about 80%, in some embodiments greater than about 85%, greater than about 90%, greater than about 95%, or greater than about 99% of all other macromolecular species present in the composition. In some embodiments, the subject material is purified to substantial homogeneity (impurity species in the composition are not detectable by conventional detection methods), wherein the composition consists essentially of a single macromolecular species.

Coelenterazine derivatives

In some embodiments, the present invention provides novel coelenterazine derivatives of formula (Ia) or (Ib):

wherein R is²Selected from the group consisting of:

or C_2-5A linear alkyl group;

R⁶selected from the group consisting of-H, -OH, -NH₂-OC (O) R or-OCH₂OC (O) R;

R⁸is selected from the group consisting of

H or lower cycloalkyl;

wherein R is³And R⁴Are both H or both C_1-2An alkyl group;

w is-NH₂Halo, -OH, -NHC (O) R, -CO₂R；

X is-S-, -O-or-NR²²-；

Y is-H, -OH OR-OR¹¹；

Z is-CH or-N-;

each R¹¹Independently is-C (O) R' or-CH₂OC(O)R”；

R²²Is H, CH₃Or CH₂CH₃；

Each R is independently C_1-7Straight chain alkyl or C_1-7A branched alkyl group;

r' is C_1-7Straight chain alkyl or C_1-7A branched alkyl group;

the bond of the dotted line indicates the presence of an optional ring, which may be saturated or unsaturated;

provided that when R is²Is composed of

When R is⁸Is not that

Provided that when R is²Is composed of

When R is⁸Is that

Or lower cycloalkyl; and

provided that when R is⁶Is NH₂When R is²Is composed of

Or C_2-5An alkyl group;

or R⁸Is not that

As used herein, the term "alkyl" pertains to a monovalent moiety obtained by withdrawing a hydrogen atom from a hydrocarbon compound, and which may be saturated, partially unsaturated, or fully unsaturated. The alkyl group may be linear or branched. Alkyl groups may be optionally substituted, for example, with halo. Examples of straight-chain alkyl radicals include, but are not limited to, ethyl, n-propyl, n-butyl and n-propyl, n-hexyl and n-heptyl. Examples of unsaturated alkyl groups having one or more carbon-carbon double bonds include, but are not limited to, vinyl (vinyl, -CH ═ CH) ₂) 2-propenyl (allyl, -CH-CH ═ CH)₂) And butenyl. Examples of unsaturated alkyl groups having one or more carbon-carbon triple bonds include, but are not limited to, ethynyl and 2-propynyl (propargyl). Examples of branched alkyl groups include isopropyl, isobutyl, sec-butyl, tert-butyl and isopentyl.

As used herein, the term "lower cycloalkyl" pertains to a monovalent moiety obtained by removing a hydrogen atom from a hydrocarbon compound having 3 to 5 carbon atoms. Examples of saturated lower cycloalkyl groups include, but are not limited to, groups such as cyclopropyl, cyclobutyl, and cyclopentyl. Examples of unsaturated lower cycloalkyl groups having one or more carbon-carbon double bonds include, but are not limited to, groups such as cyclopropenyl, cyclobutenyl, and cyclopentenyl.

As used herein, the term "halo" pertains to a halogen, such as Cl, F, Br, or I.

In some embodiments, R²Is that

And X is O or S. In other embodiments, R²Is C_2-5A linear alkyl group. In certain embodiments, R⁸Is that

Lower cycloalkyl or H. In other embodiments, R⁸Is benzyl. In some embodiments, R' is-C (CH)₃)₃、-CH(CH₃)₂、-CH₂C(CH₃)₃or-CH₂CH(CH₃)₂。

In some embodiments, the present invention provides a compound according to formula (IIa) or (IIb):

Wherein X is O or S, R⁶Is H or OH, R¹¹As defined above, and the dashed bond indicates the presence of an optional ring.

In some embodiments, the present invention provides compounds of formula (IIIa) or (IIIb):

wherein R is¹²Is C_2-5Straight-chain alkyl, furyl or thienyl, R⁶Is H or OH, R¹¹As defined above, and the dashed bond indicates the presence of an optional ring.

In some embodiments, the present invention provides compounds of formula (IVa) or (IVb):

wherein X is O or S, R⁶Is H or OH, R⁸Is H,

Or lower cycloalkyl, R³、R⁴And R¹¹As defined above, and the dotted lineThe bond (b) indicates the presence of an optional ring.

In some embodiments, the present invention provides a compound according to formula (Va) or (Vb):

wherein R is⁸Is benzyl, R¹¹As defined above, and the dashed bond indicates the presence of an optional ring.

In some embodiments, the present invention provides novel coelenterazine derivatives of formula (VIa) or (VIb):

wherein R is²Is selected from the group consisting of

Or C_2-5Straight chain alkyl groups;

R⁶selected from the group consisting of-H, -OH, -NH₂-OC (O) R or-OCH₂OC (O) R;

R⁸selected from the group consisting of:

h or lower cycloalkyl;

wherein R is³And R⁴Are both H or both C_1-2An alkyl group;

w is-NH₂Halo, -OH, -NHC (O) R, -CO ₂R；

X is-S-, -O-or-NH-;

y is-H, -OH OR-OR¹¹；

Z is-CH or-N-;

each R¹¹Independently is-C (O) R' or-CH₂OC(O)R”；

Each R is independently C_1-7Straight chain alkyl or C_1-7A branched alkyl group;

r' is C_1-7Straight chain alkyl or C_1-7A branched alkyl group;

the bond of the dotted line indicates the presence of an optional ring, which may be saturated or unsaturated;

provided that when R is²Is composed of

When R is⁸Is not that

Provided that when R is²Is composed of

When R is⁸Is that

Or lower cycloalkyl; and

provided that when R is⁶Is NH₂When R is²Is composed of

Or C_2-5An alkyl group;

or R⁸Is not that

Suitable compounds according to the invention include

Isomers, salts and protected forms

Certain compounds may exist in one or more specific geometric, optical, enantiomeric, diastereomeric, epimeric, stereoisomeric, tautomeric, conformational or anomeric forms, including, but not limited to, cis and trans; e-type and Z-type; type c, type t and type r; inward and outward; r, S and meso forms; form D and form L; type d and type l; (+) and (-) types; keto, enol, and enol forms; cis and trans; syncline and anticline forms; the alpha and beta forms; an axial form and a flat form; boat, chair, twist, envelope and half-chair; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms").

Note that, except as discussed below with respect to tautomeric forms, specifically excluded from the term "isomers" as used herein, are structural (or configurational) isomers (i.e., isomers in which the linkages between atoms differ rather than merely the spatial positions of the atoms being different). For example, methoxy, -OCH₃Should not be interpreted as referring to its structural isomers, hydroxymethyl, -CH₂And (5) OH. Similarly, reference to an o-chlorophenyl group should not be construed as a reference to its structural isomer, m-chlorophenyl. However, reference to a class of structures may include the structural isomeric forms (e.g., C) belonging to that class entirely_1-7Alkyl groups include n-propyl and isopropyl; butyl includes n-butyl, isobutyl, sec-butyl and tert-butyl; methoxyphenyl includes o-methoxyphenyl, m-methoxyphenyl, and p-methoxyphenyl).

Note that specifically included in the term "isomer" are compounds having one or more isotopic substitutions. For example, H may be in any isotopic form, including¹H、²H (D) and³h (T); c may be in any isotopic form, including¹²C、¹³C and¹⁴c; o may be in any isotopic form, including¹⁶O and¹⁸o; and the like.

Unless otherwise indicated, reference to a particular compound includes all such isomeric forms, including (in whole or in part) racemates and other mixtures thereof. Methods for preparing (e.g., asymmetric synthesis) and separating (e.g., fractional crystallization and chromatography methods) such isomeric forms are known in the art or are readily obtainable by modifying the methods taught herein or known methods in known ways.

Unless otherwise specified, reference to a particular compound also includes ionic, salt, solvate, and protected forms thereof, e.g., as discussed below. It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the active compound, e.g., a pharmaceutically acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge et al, J.pharm.Sci.,66:1-19 (1977).

For example, if the compound is anionic, or has a functional group that can be anionic (e.g., -COOH can be-COO-), a salt can be formed using a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na⁺And K⁺Alkaline earth cations such as Ca²⁺And Mg²⁺And other anions such as Al³⁺. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH)⁴⁺) And substituted ammonium ions (e.g., NH)₃R⁺、NH₂R₂ ⁺、NHR₃ ⁺、NR₄ ⁺). Some examples of suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, benzylaniline, choline, meglumine and tromethamine, and amino acids such as lysine and arginine. A common example of a quaternary ammonium is N (CH) ₃)₄ ⁺。

If the compound is cationic, or has a functional group which may be cationic (e.g., -NH)₂May be-NH₃ ⁺) Salts can be formed using suitable anions. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acidAcids, phosphoric acid and phosphorous acid. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: acetic, propionic, succinic, glycolic, stearic, palmitic, lactic, malic, pamoic, tartaric, citric, gluconic, ascorbic, maleic, hydroxymaleic, phenylacetic, glutamic, aspartic, benzoic, cinnamic, pyruvic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, benzenesulfonic, p-toluenesulfonic, methanesulfonic, ethanesulfonic, ethanedisulfonic, oxalic, pantothenic, isethionic, valeric, lactobionic and gluconic acids. Examples of suitable polymeric anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

It may be convenient or desirable to prepare, purify, and/or handle the corresponding salts of the active compounds. The term "solvate" as used herein in its ordinary sense refers to a complex of a solute (e.g., active compound, salt of active compound) and a solvent. If the solvent is water, the solvent may be conveniently referred to as a hydrate, e.g., a monohydrate, dihydrate, trihydrate, and the like.

It may be convenient or desirable to prepare, purify, and handle the active compound in chemically protected form. The term "chemically protected form" as used herein pertains to a compound in which one or more reactive functional groups are protected from undesired chemical reactions, i.e. are protecting groups (also referred to as masking or blocking groups). By protecting the reactive functional group, reactions involving other non-protected reactive functional groups can be carried out without affecting the protecting group; the protecting group can generally be removed in a subsequent step without substantially affecting the rest of the molecule. See, for example, Protective Groups in Organic Synthesis (T.Green and P.Wuts, Wiley, 1999).

For example, a hydroxy group may be protected as an ether (-OR) OR ester (-OC (═ O) R), for example, as: tert-butyl ether; benzyl ether, benzhydryl (diphenylmethyl) ether or trityl (triphenylmethyl) ether; trimethylsilyl ether or tert-butylmethylsilyl ether; or B Acyl ester (-OC (═ O) CH₃-OAc). For example, aldehyde or ketone groups may be protected as acetals or ketals, respectively, in which a carbonyl group(s) is/are reacted by reaction with, for example, a primary alcohol>Conversion of C ═ O) to diether(s) ((s)>C(OR)₂). The aldehyde or ketone group is easily regenerated by hydrolysis with an excess of water in the presence of an acid. For example, amine groups may be protected as, for example, amides or urethanes, for example, as: methylamide (-NHCO-CH)₃) (ii) a Benzyloxyamide (-NHCO-OCH)₂C₆H₅-NHCbz); as tert-butoxyamide (-NHCO-OC (CH))₃)₃-NH-Boc); 2-Biphenyl-2-propoxyamide (-NHCO-OC (CH)₃)₂C₆H₄C₆H₅-NH-Bpoc) as 9-fluorenylmethoxyamide (-NH-Fmoc), as 6-nitroveratryloxyamide (-NH-Nvoc), as 2-trimethylsiloxyethylamide (-NH-Teoc), as 2,2, 2-trichloroethoxyamide (-NH-Troc), as allyloxyamide (-NH-Alloc), as 2- (phenylsulfonyl) ethoxyamide (-NH-Psec); or, where appropriate, as an N-oxide.

For example, the carboxylic acid group may be protected as an ester, for example, as: c_1-7Alkyl esters (e.g., methyl esters; tert-butyl esters); c_1-7Haloalkyl esters (e.g. C)_1-7Trihaloalkyl esters); three C _1-7alkylsilyl-C_1-7An alkyl ester; or C_5-20aryl-C_1-7Alkyl esters (e.g., benzyl esters; nitrobenzyl esters); or as an amide, for example, as a methylamide.

For example, a thiol group may be protected as a thioether (-SR), for example, as: benzyl sulfide; acetamidomethyl methyl ether (-S-CH)₂NHC(＝O)CH₃)。

Synthesis of coelenterazine derivatives

Coelenterazine derivatives according to the invention may be synthesized according to those methods described in detail in examples 1-16.

Mutant oplophorus luciferase

In embodiments of the invention, various techniques described herein are used to identify the sites of amino acid substitutions to produce improved synthetic OgLuc polypeptides. Additional techniques are used to optimize codons in polynucleotides encoding different polypeptides to enhance expression of the polypeptides. It was found that making one or more amino acid substitutions, alone or in different combinations, results in a synthetic OgLuc-type polypeptide with enhanced luminescence (e.g., enhanced brightness, enhanced signal stability, enhanced enzyme stability, and/or changes in relative substrate specificity). In addition, including one or more codon-optimized substitutions in the polynucleotides encoding the different synthetic OgLuc variant polypeptides results in enhanced expression of the polypeptides in different eukaryotic and prokaryotic expression systems. One embodiment of the invention is a polynucleotide encoding a synthetic OgLuc variant polypeptide that is soluble when expressed in prokaryotic and/or eukaryotic cells and is active in monomeric form.

The OgLuc variants of the invention can be coupled to any protein of interest or molecule of interest. In some embodiments, the variants are fusion proteins, e.g., some variants with an N-terminal or C-terminal attachment

Polypeptide coupling. Unless otherwise indicated, as

Variants of the fusion include "HT 7" as part of its name, e.g., "IVY-HT 7". In some embodiments, a signal sequence (e.g., a naturally occurring Cereus tenuis signal sequence) is attached to the N-terminus of the fusion protein to facilitate secretion of the fusion protein from the cell. Signal sequences, other than those naturally occurring with OgLuc luciferase, are known in the art to facilitate protein secretion in mammalian cells or other cell types. The signal sequence in combination with the membrane anchor sequence can be used to localize or display the OgLuc variant on the exterior surface of the cell membrane. Other methods known in the art can also be used to localize the OgLuc variant to other locations in the membrane or cell.

In some embodiments, the invention provides modified decapod luciferases with enhanced luminescence relative to the corresponding parent variant decapod luciferases. For example, the variant OgLuc of the parent is C1+ A4E, IVY, IV, QC27, QC27-9a, 9B8, 9B8 opt + K33N, 9B8 opt + K33N +170G, V2, or "L27V". In another embodiment, the present invention provides modified decapod luciferases utilising a novel coelenterazine. In one embodiment, the modified decapod luciferase has an alteration in the relative specificity for a native, known or novel coelenterazine. In one embodiment, the modified decapod luciferase has a relative specificity change relative to a corresponding parent variant decapod luciferase.

In some embodiments, the corresponding parent variant decapod luciferase is a decapod species, including different species from the decapod family of subjects, including, but not limited to, the following luciferases: the family aenopsis (Aristeidae), including the short-limb paraphrenia (Plesiopenaeus ruduscans); pandalidea family, including Allophyllus spp (Heterocarpus spp.) and Parapanadalus richardi, Cymbidae (Solenoceridae), including Hymenopenaeus bideis and Tropical metapenaeus chinensis (Mesopenaeus tropicalis); the family of the fireflies (Luciferidae), including positive-type fireflies (Lucifer typus); sergestidae (Sergestidae) including Charophytus maxima (Sergestes atlanticus), Scophytus arctica (Sergestes arcticus), Cyanea acuta (Sergestes armatus), Sergestes pedformis (Sergestes armenius), Sergestes cornutus, Sergestes edwards, Sergestes hensenensis, Cydonia pectinifera (Sergestes pectus), Sergestes sargi, Cydonia formosanus (Sergestes sementis), Sergestis sempervirgula, Sergestissa charliensis, Sergestis grandis, Sergestis cerasus (Sergestis lucens), Sergestis ruber (Sergestis), Sergestis punctilex, Sergestissa robusta, Sergestis scintillation cerasus (Sergestis), and Sergestella; the family of glassshrimp (Pasiphaeidae) including Glyphus marsuphialis, Paecilomyces argenteus (Leptochlea bermudensis), Gouqua glaucus (Parapasiphae sulcations), and Pasiphea tarda; acetoceridae (Oplophorridae) including Acantophyta acanthis, Acantophyta acutiferrons, Acantophyta brevialis, Acantophyta cuulata, short-angled Acanthopanax acarus (Acantophyta curitoris), abnormal Acanthopanax acarus (Acantophyta eximia), Acantophyta gracilis, Acantophyta kinensis, Acantophyta kuwana, Acantophyta medula, Acantophyta microphylla, Acantophyta microphyllata, Acantophyta microphylla, Acantophyta pelagica, Acantophyta propriocera, Acanthophyta purpurea, Acantha haematoptera, Acantha sanguinea, Acanthophyta sanguinea, Acanthus serophyra, Acanthus macrobrachium, Acanthus giganteus, Acanthus macrobrachium, Ephyllus macrobrachium (Eutropha), Acophyta macrobrachium, Eutropha macrobrachium, Euonyx japonicus, Euonymus (Euonymus), Euonymus macrobrachium, Euonymus macrobrachium, Euonymus), Euonymus macrobrachium, Euonymus (Euonymus), Euonymus macrobrachium, Euonymus (Euonymus), Euonymus macrobrachium macro; and the family Penaeidae (Thalassociaceae), including Strongylocentrotus spinosus (Chlorotocoids spinouda), Stenopsis chinensis (Thalassocias crinita) and Thalassocias lucida. In some embodiments, the modified luciferase has an increased luminescence emission, e.g., at least 1.3-fold, at least 2-fold, or at least 4-fold, in prokaryotic and/or eukaryotic cells relative to a corresponding wild-type luciferase. In some embodiments, one or more properties of the modified decapod luciferase are compared to similar properties of a luciferase from another species (e.g., a firefly luciferase or a renilla luciferase).

In some embodiments, the OgLuc variant has at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, or 100% amino acid sequence identity to SEQ ID No. 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 27, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 56, 58, 60, 62, 64, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, or 95. In some embodiments, the OgLuc variant, or a functional fragment thereof, has no more than 5 differences, or more preferably, no more than 4, 3, 2, or 1 differences, or most preferably no differences, wherein the differences occur in positions corresponding to mode positions 1, 2, 3, 5, 8, 10, 12, 14, 15, 17, or 18 according to formula (VII) of table 4. The differences may also include gaps or insertions between the mode positions of table 4.

In some embodiments, the OgLuc variants of the invention have one or more heterologous amino acid sequences (fusion polypeptides such as fusion polypeptides having an epitope or fusion tag) at the N-terminus, C-terminus, or both, which optionally interact directly or indirectly with the molecule of interest. In some embodiments, the presence of the heterologous sequence does not substantially alter the luminescence of the OgLuc variant prior to or after interaction with the molecule of interest. In some embodiments, the heterologous amino acid sequence is an epitope tag. In some embodiments, the heterologous amino acid sequence is such that, during or after interaction with the molecule of interest, it undergoes a conformational change which, in turn, alters the activity of the OgLuc variant, e.g., an OgLuc variant having such an amino acid sequence is useful for detecting allosteric interactions. The OgLuc variant or a fusion with the OgLuc variant or a fragment thereof can be used as a reporter.

In some embodiments, a fragment of an OgLuc variant of the invention is fused to a heterologous amino acid sequence, thereby forming a β -barrel, the fusion protein being capable of generating luminescence from a naturally occurring coelenterazine or analogs thereof, including various known coelenterazines discussed herein, or a novel coelenterazine of the invention.

Also provided are polynucleotides encoding the OgLuc variants or fusions thereof of the invention, isolated host cells having the polynucleotides or OgLuc variants or fusions thereof, and methods of using the polynucleotides, OgLuc variants or fusions or host cells of the invention.

The term "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more series or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence across a comparison window or designated region, as measured using any number of sequence comparison algorithms or by manual alignment and visual inspection. Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be performed by: smith et al, (J.mol.biol. 147:195-197(1981)), homology alignment algorithms of Needleman and Wunsch, (J.mol.biol., 48:443-453(1970)), similarity search methods of Pearson and Lipman, (Proc.Natl.Acad.Sci.USA, 85:2444-2448(1988)), computerized implementations of algorithms, such as FASTA, SSEARCH, GGSEARCH (available from the TA server of William R.Pearson, university of Giffy as http:// fa.bio.virginia.edu/fa _ www int 2/fa _ stm. shtml), Clustal series programs (Chenta et al, Nucl. Acsis Res.31(13): 3497): 2003/www.ebi.ac.uk), or other sequences available from the software of Hasta et al, SEQ ID No. Across.: 2003/www.ebi.ac.uk, or www.ch.embnet.org. It is known in the art that generating alignments with maximum identity between polypeptide sequences with significant sequence changes may involve the use of professional methods, such as ABA methods (Raphael et al, Genome Res.14(11):2336-2346(2004)), other suitable methods, or alignments using two linked identical copies of a polypeptide sequence.

The term "nucleic acid molecule", "polynucleotide" or "nucleic acid sequence" as used herein refers to a nucleic acid, including DNA or RNA, that includes the coding efficiency required to produce a polypeptide or protein precursor. The encoded polypeptide can be a full-length polypeptide, a fragment thereof (less than full-length), or a fusion polypeptide produced by fusion of a full-length polypeptide or a fragment thereof with another polypeptide.

A polynucleotide encoding a protein or polypeptide means a nucleic acid sequence that includes a coding region of a gene, or in other words, a nucleic acid sequence that encodes a gene product. The coding region may be present in cDNA, genomic DNA or RNA form. When present in DNA form, the oligonucleotide may be single-stranded (e.g., the sense strand) or double-stranded. If desired, appropriate control elements such as enhancers/promoters, splice junctions, polyadenylation signals, and the like may be located adjacent to the coding region of the gene to allow transcription of the primary RNA transcript and/or proper initiation of proper processing. Other control or regulatory elements include, but are not limited to, transcription factor binding sites, splicing signals, polyadenylation signals, termination signals, and enhancer elements.

"peptide," "protein," and "polypeptide" mean chains of amino acids of different lengths, regardless of post-translational modification (e.g., glycosylation or phosphorylation). The nucleic acid molecules of the invention encode variants of artificial (i.e., synthetic) variant proteins or polypeptide fragments thereof that have at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to the amino acid sequence of the parent protein from which they are derived, which may be a naturally occurring (natural or wild-type) sequence or a subsequently further modified variant sequence. The term "fusion polypeptide" or "fusion protein" refers to a chimeric protein comprising a reference protein (e.g., an OgLuc variant) linked at the N-terminus and/or C-terminus to one or more heterologous sequences (e.g., a non-OgLuc polypeptide). Heterologous sequences can include, but are not limited to, reporter proteins such as

Fusion proteins (Promega Corp.), FlAsH (fluorescein containing arsenic helix binder), and reaash (red containing arsenic helix binder) (e.g., LUMIO)^TMTag recognition sequences (Invitrogen)), Chloramphenicol Acetyltransferase (CAT), β -galactosidase (β -Gal), lactamase (P-Gal), neomycin resistance (Neo), GUS, glucopyranosyl galactose, Green Fluorescent Protein (GFP), luciferase (e.g., Renilla reniformis) luciferase, firefly luciferase (e.g., Photinus pyralis or Photuris pensylvanica) or click luciferase (e.g., jamaicus click beetles or pyrexius terrestris) or firefly (glowworm) luciferase (e.g., phothrix hirtanus), xylosidase, thymidine kinase, arabinosidase and SNAP tags, CLIP tags, ACP tags and MCP tags (New glaucas). In one embodiment, the chimeric protein comprises a peptide at the N-terminus

Fusion protein (Promega Corp.) linked OgLuc variants. In another embodiment, the chimeric protein comprises a peptide linked at the C-terminus to a peptide linker

Fusion protein-linked OgLuc variants.

Nucleic acids are known to contain different types of "mutations," meaning changes at specific base positions in the sequence of nucleotides relative to the wild-type sequence. Mutation may also refer to the insertion or deletion of one or more bases to make the nucleic acid sequence different from the reference, e.g., a wild-type sequence, or substitution with a stop codon. "substitution" refers to a change in the amino acid at a particular position in the sequence, for example, from A to E at position 4.

The term "vector" refers to a nucleic acid molecule into which a fragment of DNA can be inserted or cloned, which can be used to transfer a DNA segment into a cell and which is capable of replication in the cell. The vector may be derived from a plasmid, phage, virus, cosmid, and the like.

As used herein, the term "wild-type" or "native" refers to a gene or gene product having the characteristics of a gene or gene product isolated from a naturally occurring source. Wild-type genes are the "wild-type" forms that are most commonly observed in the population and are thus arbitrarily designated genes. Conversely, the term "mutant" refers to a gene or gene product that exhibits a modification (i.e., altered characteristic) in sequence and/or functional properties when compared to the wild-type gene or gene product. It is noted that naturally occurring mutants can be isolated; these are identified by the fact that they have altered properties when compared to the wild-type gene or gene product.

Exemplary polynucleotides and proteins

The present invention includes OgLuc variants or protein fragments thereof having at least one amino acid substitution relative to wild-type OgLuc, e.g., those having a deletion, e.g., a deletion of 1 to about 5 residues, and chimeras (fusions) thereof (see U.S. patent publication No. 2009/0253131 and WIPO publication No. WO 2007/120522, the disclosures of which are incorporated herein by reference), which substitution results in an OgLuc variant having enhanced stability, enhanced luminescence, e.g., increased luminescence emission, greater photodynamic stability, and/or altered luminescence color. The sequence of the OgLuc variant is substantially identical to the amino acid sequence of the corresponding wild-type OgLuc. A polypeptide or peptide having a substantially identical sequence means that the amino acid sequence is to a large extent, but not completely, identical and retains the functional activity of the sequence with which it is associated. Typically, two amino acid sequences are substantially identical if they are at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%, but less than 100% identical in amino acid sequence. In some embodiments, the OgLuc variants are encoded by recombinant polynucleotides. In some embodiments, the OgLuc variant, or a functional fragment thereof, has no more than 5 differences, or more preferably no more than 4, 3, 2, or 1 differences, or most preferably no differences, wherein the differences occur at mode positions 1, 2, 3, 5, 8, 10, 12, 14, 15, 17, or 18 corresponding to formula (VII) according to table 4. The differences may also include gaps, insertions, or swaps between the mode positions of table 4.

The OgLuc variant or fusion proteins of the invention can be prepared by recombinant methods or by solid phase chemical peptide synthesis methods. Such methods are known in the art.

Methods of use and kits

The compounds and proteins of the invention may be used in any method where luciferase and a luciferase substrate, e.g., coelenterazine, have been used. For example, it may be used in a bioluminescent method using an analogue of coelenterazine to detect one or more molecules in a sample, e.g., an enzyme, a cofactor for an enzymatic reaction, an enzyme substrate, an enzyme inhibitor, an enzyme activator, or an OH group, or one or more conditions, e.g., redox conditions. The sample can include an animal (e.g., a vertebrate), a plant, a fungus, a physiological solution (e.g., blood, plasma, urine, mucus secretions, and similar physiological solutions), a cell lysate, a cell supernatant, or a purified cell fraction (e.g., a subcellular fraction). The presence, amount, spectral distribution, emission kinetics or specific activity of such molecules can be detected or quantified. Molecules can be detected or quantified in solutions including multiphase solutions (e.g., emulsions or suspensions) or solid supports (e.g., particles, capillaries, or detection vessels). In some embodiments, the OgLuc variants can be used in luminescence-based assays to detect enzymes of interest, e.g., CYP450 enzymes, MAO a or B enzymes, caspases, and the like. The novel coelenterazine may be used with photoproteins such as aequorin, hydroid or iPhotina. In some embodiments, the OgLuc variant can be used as an energy donor for another molecule (e.g., a fluorophore, a chromophore, or a nanoparticle).

The invention also provides polynucleotides encoding transcription reporters. In some embodiments, the OgLuc variant or fragment thereof can be operably linked to transcriptional regulatory sequences (e.g., one or more enhancers, promoters, transcriptional termination sequences, or combinations thereof) to form an expression cassette. For example, the OgLuc variant can be operably linked to a minimal promoter and cAMP-responsive element (CRE).

The proteins of the invention can be used as biological sensors, e.g., OgLuc variants that have one or more altered activities in the presence of another molecule (e.g., one or more molecules of interest), or under certain conditions. Upon interaction with a molecule of interest or under certain conditions, the biological sensor undergoes a conformational or chemical change, resulting in a change in the enzymatic activity of the luminescence, e.g., specific activity, spectral distribution, or emission kinetics. For example, an OgLuc variant of the invention, e.g., a circularly permuted variant, can include a region that interacts with a molecule of interest. Alternatively, for example, the OgLuc variant can be coupled to an energy receptor, e.g., a fluorescent protein, and include an interaction region that alters the efficiency of energy transfer from the enzyme to the energy receptor. For example, biological sensors can be generated by inserting appropriate sensor regions into the OgLuc variant sequence to detect proteases, kinases, ligands, binding proteins such as antibodies, cyclic nucleotides such as cAMP or cGMP, or metals such as calcium. One or more sensor regions may be inserted at the C-terminus, N-terminus, and/or at one or more suitable positions of the polypeptide sequence, wherein the sensor region comprises one or more amino acids. In the case of circularly permuted OgLuc variants, a receptor region can be inserted between the N-terminus and the C-terminus of the parent OgLuc variant. In addition, one or all of the inserted receptor regions may include linker amino acids to couple the receptors to the rest of the OgLuc variant. Examples of luciferase biosensors are disclosed in U.S. patent application publication nos. 2005/0153310 and 2009/0305280 and PCT publication No. WO 2007/120522 a2, each of which is incorporated herein by reference.

In various embodiments, the OgLuc variants disclosed herein can be used to transfer energy (e.g., in a Bioluminescence Resonance Energy Transfer (BRET) assay) to an energy receptor. For example, the OgLuc variants used in BRET assays can be used to determine whether two molecules are able to bind or co-localize to each other in a cell. For example, the OgLuc variant can be used as a bioluminescent donor molecule in combination with a molecule or protein of interest to construct a first fusion protein. In various embodiments, the first fusion protein comprises an OgLuc variant and a protein of interest. In various embodiments, the first fusion protein comprising the OgLuc variant can be used in BRET assays to detect protein/protein interactions in systems including, but not limited to, cell lysates, intact cells, and live animals. In a different embodiment of the process according to the invention,

can be used as fluorescent acceptor molecules. In some embodiments of the present invention, the substrate is,

can be fused to a second protein of interest or an OgLuc variant. For example, the OgLuc variant can be combined with

Fusion, expression in cells or animals, and use of fluorescence

Ligands such as

And labeling by using TMR ligand. The fusion can then be stimulated to fluoresce in the presence of the cell-permeabilized OgLuc substrate. In some embodiments, BRET may be performed using OgLuc variants in combination with fluorescent proteins, including but not limited to Green Fluorescent Protein (GFP), or Red Fluorescent Protein (RFP), or fluorescent labels including fluorescein, rhodamine green, oregon green, or Alexa 488, to name a few non-limiting examples.

In various embodiments, the OgLuc variants and novel coelenterazines of the present invention can be used in Protein Complementation Assays (PCA) to detect the interaction of two biomolecules, e.g., polypeptides. For example, the OgLuc variants of the invention can be separated into two fragments at sites that allow for separation, and each fragment of the isolated OgLuc variants can be fused to one of a pair of polypeptides of interest (e.g., FKBP and FRB) that are believed to interact. If the two polypeptides of interest do in fact interact, the OgLuc fragments enter into proximity to each other to reconstitute a functional, active OgLuc variant. In some embodiments, the activity of the reconstituted OgLuc variants can be subsequently detected and measured using natural or known coelenterazine or the novel coelenterazines of the invention. In some embodiments, the cleaved OgLuc variants can be used in a more general complementation system similar to lac-Z (Langley et al, PNAS 72:1254-1257(1975)) or ribonuclease S (Levit and Berger, J.biol. chem.251: 1333-1339 (1976)). In some embodiments, an OgLuc variant fragment (designated "a") known to be complementary to another OgLuc variant fragment (designated "B") can be fused to a target protein, and the resulting fusion can be monitored via luminescence in a cell or cell lysate comprising fragment B. In some embodiments, the source of fragment B may be the same cell (e.g., if the gene of fragment B is inserted into the genome of the cell or on another plasmid contained in the cell) or it may be a lysate or purified protein derived from another cell. In some embodiments In (B), fragment B and polypeptides capable of being attached to a solid support such as

To capture or immobilize the same fusion protein (fragment a). In some embodiments, luminescence may be used to demonstrate successful capture or to quantify the amount of captured material.

In various embodiments, the OgLuc variants and/or novel coelenterazines of the present invention can be used to quantify coelenterazines. In some embodiments, coelenterazine (e.g., native or known coelenterazine, or the novel coelenterazines of the present invention) may be used as a probe for specific biochemical activities such as apoptosis and drug metabolism. In some embodiments, coelenterazine concentration may be coupled to a particular enzyme activity by a "precolumn" or "pro-substrate" to which a particular enzyme of interest is capable of acting. In some embodiments, the proventriculin is a molecule that when combined with luciferase cannot directly support luminescence but can be converted to coelenterazine through catalytic processing by the particular enzyme of interest. In some embodiments, methods such as those used in drug metabolism can be used for enzymes, e.g., cytochrome P450 enzymes, monoamine oxidases, and glutathione S transferases; and apoptosis, such as caspases. For example, coelenterazine (e.g., native or known coelenterazine, or the novel coelenterazines of the present invention) may be modified to include a cleavable group, such as 6' -O-methyl. In some embodiments, when incubated with a particular cytochrome P450 enzyme, the 6' O-methyl group is cleaved and the prodromycin is converted to coelenterazine that can be detected using the OgLuc variants of the invention. In some embodiments, the proventriculin can be combined with other components (e.g., photoproteins such as the OgLuc variants of the invention) necessary to support luminescence to provide a single reagent and homogeneous assay. For example, when a reagent is added to a sample, luminescence is generated as the pre-coelenterazine is converted to coelenterazine. In various embodiments, similar assays may be developed for other enzymes, small molecules, or other cellular processes that may be associated with the production of coelenterazine from pre-coelenterazine.

In some embodiments, the OgLuc variants and/or novel coelenterazines of the present invention can be used as genetic transcription reporter systems. In some embodiments, the OgLuc variant can be compared to a luciferase that emits light at a different wavelength (e.g., a red click beetle luciferase (CHROMA-LUC)^TM(ii) a Promega Corp.)) multiple complexing. For example, if the OgLuc variant of the invention is used as a functional reporter, a red CHROMA-LUC^TMLuciferase can be used to control non-specific effects on genetic regulation or to normalize for transfection efficiency. In some embodiments, resolution from the OgLuc variant (about 460nm) and red CHROMA-LUC can be readily achieved using a photometer with a wavelength-discriminating filter^TM(about 610nm) to enable the measurement of both signals from the same sample. In another example, the OgLuc variants of the invention can be used as transcription reporters and paired with luciferases that emit at different wavelengths contained in the assay reagents. For example, the OgLuc variants of the invention can be used as transcription reporters and paired with aequorin or firefly luciferase biosensors that are cyclically exchanged for cAMP, or both, to detect multiple pathways in a single sample. In this system, for example, aequorin can be used to detect and/or measure calcium, a biosensor to detect and/or measure cAMP, and an OgLuc variant to monitor downstream gene expression. In another example, the OgLuc variant can be used with one or more additional luciferases, where the luminescence of each luciferase can be measured separately by using selective enzyme inhibitors. For example, the luminescence of a first luciferase may be measured after addition of appropriate substrates and buffers, and then a second luciferase measured after subsequent addition of appropriate substrates and buffers and one or more inhibitors selective for the first luciferase. In another example, luciferase contained in the assay reagent can be used to measure a particular aspect of cell physiology, e.g., measuring ATP to assess cell viability, or measuring caspase activity to assess apoptosis.

In various embodiments, the OgLuc variants of the invention are used as reporters in difficult to transfect cell lines or possibly even in primary cells that do not divide (e.g., stem cells or HepG2 cells). Due to its high signal intensity, the OgLuc variants of the invention will be able to make luminescence detectable when transfection efficiency is low. In some embodiments, the OgLuc variant can be used as a reporter in a cell that is particularly sensitive to conditions associated with transfection (e.g., it is sensitive to increased DNA concentrations or to the addition of transfection reagents). Thus, in various embodiments, due to the enhanced luminescence of the OgLuc variants of the invention, sufficient levels of luminescence can be achieved with reduced toxic burden on sensitive cells using lower DNA concentrations, less transfection reagent, and/or shorter post-transfection time before the start of the assay. In various embodiments, the enhanced luminescence of the OgLuc variant allows for the detection of a signal at a later point in time. In still further embodiments, the OgLuc variant can be used as a reporter for a single copy of the native promoter.

In various embodiments, the OgLuc variants of the invention can be used as fusion tags for target proteins of interest as a way to monitor intracellular levels of the target protein. In some embodiments, OgLuc variants can be used to monitor specific proteins involved in stress response pathways (e.g., DNA damage, oxidative stress, inflammation) in cells, as a way to explore the roles different types of stimuli may play in these pathways. In some embodiments, the OgLuc variants can also be used as a tool to monitor cell trafficking of a target protein. For example, the OgLuc variants can also be fused to viral genomes (e.g., HIV, HCV) so that titer levels, i.e., infectivity, can be monitored in cells after treatment with an effective antiviral agent. In some embodiments, the variant may also be conjugated to a Green Fluorescent Protein (GFP) or

Fusion (except for target protein) for Fluorescence Activated Cell Sorting (FACS) to identify high expressing clones.

In various embodiments, the enhanced signaling of the OgLuc variants and the small size of the OgLuc gene can facilitate the identification of robust, stable cell lines that express cytoplasmic or secreted forms of the OgLuc variants of the invention. The relatively small gene sequences can reduce the likelihood of gene instability caused by integration of foreign DNA into the genome of the cell.

In various embodiments, the OgLuc variants of the invention can be integrated into a variety of different immunoassay concepts. For example, the OgLuc variants can be fused to a primary or secondary antibody to provide a detection method for a particular analyte. As another example, the OgLuc variant can be fused to protein a or protein G, and the fusion can then be used to detect a particular antibody that binds to a particular analyte. As another example, the OgLuc variants can bind streptavidin and be used to detect specific biotinylated antibodies that bind to specific analytes. As yet another example, complementary fragments of the OgLuc variant can be fused to a primary antibody and a secondary antibody, wherein the primary antibody recognizes a particular analyte, and the secondary antibody recognizes the primary antibody. In some embodiments, the OgLuc variant activity will reconstitute in the presence of the analyte. As yet another example, the OgLuc variant can be conjugated to an analyte (e.g., a prostaglandin) and used in a competitive trimmings ELISA format. The OgLuc variants conjugated to an analyte can also be used to detect antibodies capable of binding to the analyte, where the binding activity allows the OgLuc variants to be selectively linked to the antibody. An example of the use of Renilla luciferase for quantitatively measuring patient antibody titer to an antigen target is the luciferase co-immunoprecipitation system (Burbelo et al, Expert Review of Vaccines 9(6):567-578 (2010)).

In various embodiments, the OgLuc variants and novel substrates of the invention can be used to detect luminescence in living cells. In some embodiments, the OgLuc variant can be expressed in the cell (as a reporter or otherwise) and the cell is treated with coelenterazine (e.g., a novel coelenterazine, such as PBI-3939) that will permeate the cell in culture, react with the OgLuc variant and generate luminescence. In addition to being a cell penetrating substance, PBI-3939 showed biocompatibility with respect to cell viability comparable to native coelenterazine. In some embodiments, a form comprising chemically modified PBI-3939 known to improve the stability of native coelenterazine in media can be synthesized and used for more robust, viable cell, OgLuc variant-based reporter assays. In yet further embodiments, samples (including cells, tissues, animals, etc.) comprising the OgLuc variants and/or novel coelenterazine of the invention can be assayed using different microscopy and imaging techniques. In yet some further embodiments, the secreted OgLuc variant is expressed in a cell as part of a living cell reporter system.

In various embodiments, the OgLuc variants and/or novel coelenterazine disclosed herein can be provided as part of a kit. The kit may include one or different OgLuc variants (in the form of a polypeptide, a polynucleotide, or both) and/or coelenterazine as disclosed herein, along with suitable reagents and instructions enabling a user to perform assays such as those disclosed herein. Coelenterazine may be any of the natural, known or novel coelenterazines disclosed herein. The kit may also include one or more buffers, such as those disclosed herein.

Vectors and host cells encoding modified luciferases and fusions thereof

Once the desired nucleic acid molecule encoding the OgLuc variant or a fragment thereof (such as a fragment that is luminescent or can be complementary to another molecule to result in luminescent activity, or a luminescent fusion thereof) is prepared, an expression cassette encoding the OgLuc variant or a fragment thereof (e.g., a fragment for complementation or a luminescent fusion thereof) can be prepared. For example, a nucleic acid molecule comprising a nucleic acid sequence encoding an OgLuc variant is operably linked to a transcriptional regulatory sequence (e.g., one or more enhancers, promoters, transcriptional termination sequences, or combinations thereof) to form an expression cassette. The nucleic acid molecule or expression cassette may be introduced into a vector, e.g., a plasmid or viral vector, which optionally includes a selectable marker gene, and a vector introduced into a cell of interest, e.g., prokaryotic cells such as escherichia coli, Streptomyces spp, Bacillus spp, Staphylococcus spp, and the like, and eukaryotic cells including plants (dicotyledons or monocotyledonous plants), fungi including yeasts, e.g., Pichia (Pichia), Saccharomyces cerevisiae (Saccharomyces), or Schizosaccharomyces (Schizosaccharomyces), or mammalian cells, the lysates of which may be introduced in vitro into a transcription/translation mixture. Mammalian cells include, but are not limited to, bovine, goat, ovine, canine, feline, non-human primate cells, such as simian cells and human cells. Mammalian cell lines include, but are not limited to, CHO, COS, HEK293, HeLa, CV-1, SH-SY5Y, and NIH 3T3 cells, although a wide variety of other cell lines may also be used.

Expression of the encoded OgLuc variant can be controlled by any promoter, including synthetic promoters, capable of expression in prokaryotic or eukaryotic cells. Prokaryotic promoters include, but are not limited to, SP6, T7, T5, tac, bla, trp, gal, lac, or maltose promoters, including any fragment having promoter activity. Eukaryotic promoters include, but are not limited to, constitutive promoters, e.g., viral promoters such as CMV, SV40 and RSV promoters, as well as regulatable promoters, e.g., inducible or repressible promoters such as the tet promoter, hsp70 promoter, and synthetic promoters regulated by CRE, including any fragment having promoter activity. Expression of the encoded OgLuc variant can also be controlled by post-transcriptional programs, such as programs regulated by regulation of RNA processing or translation, e.g., by RNAi, miRNA, shRNA, siRNA, or by RNA or protein degradation. The nucleic acid molecules, expression cassettes, and/or vectors of the invention can be introduced into cells by any method, including, but not limited to, calcium-mediated transformation, electroporation, microinjection, lipofection, and the like.

Optimized sequences and vectors and host cells encoding OgLuc variants

Also provided are isolated nucleic acid molecules (polynucleotides) comprising a nucleic acid sequence encoding an OgLuc variant, a functional fragment thereof, or a fusion protein thereof of the invention. In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid sequence optimized for expression in at least one selected host. Optimized sequences include codon optimized sequences, i.e., codons that are used more frequently in one organism relative to another organism (e.g., a distantly related organism), as well as modifications to add or modify Kozak sequences and/or introns, and/or to remove unwanted sequences, e.g., potential transcription factor binding sites. Such optimized sequences may provide enhanced expression, e.g., increased levels of protein expression, when introduced into a host cell. Examples of optimized sequences are disclosed in U.S. patent No. 7,728,118 and U.S. patent application publication nos. 2008/0070299, 2008/0090291 and 2006/0068395, each of which is incorporated herein by reference.

In some embodiments, the polynucleotide comprises a nucleic acid sequence encoding an OgLuc variant of the invention that is optimized for expression in a mammalian host cell. In some embodiments, the optimized polynucleotide no longer hybridizes to the corresponding non-optimized sequence, e.g., does not hybridize to the non-optimized sequence under moderately or highly stringent conditions. The term "stringent" is used to refer to conditions of temperature, ionic strength, and the presence of other compounds under which nucleic acid hybridization is performed. Under "highly stringent" conditions, nucleic acid base pairing will only occur between nucleic acid fragments having a high frequency of complementary base sequences. Thus, when it is desired that the nucleic acids do not hybridize or anneal together with complete complementarity to each other, conditions of "moderate" or "low" stringency are often used. It is well known in the art that a large number of equivalent conditions can be used to create conditions of moderate or low stringency.

In some embodiments, the polynucleotide has at least 90%, e.g., less than 80% nucleic acid sequence identity to a corresponding non-optimized sequence, and optionally encodes at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the polypeptide encoded by the non-optimized sequence. Also provided are constructs (e.g., expression cassettes and vectors) comprising the isolated nucleic acid molecules (e.g., having optimized nucleic acid sequences), and kits comprising the isolated nucleic acid molecules, constructs, or vectors.

A nucleic acid molecule comprising a nucleic acid sequence encoding an OgLuc variant of the invention, a fragment thereof, or a fusion thereof, is optionally optimized for expression in a particular host cell, and is also optionally operably linked to transcriptional regulatory sequences, such as one or more enhancers, promoters, transcriptional termination sequences, or combinations thereof, to form an expression cassette.

In some embodiments, the nucleic acid sequence encoding the OgLuc variants, fragments thereof, or fusions thereof of the invention are optimized by replacing codons, e.g., at least 25% of the codons in the parent OgLuc sequence are replaced with codons that are preferentially used in the particular (selected) cell. Preferred codons have a relatively high codon usage frequency in the selected cell, and preferably their introduction results in relatively few transcription factor binding sites for transcription factors present in the selected host cell, and relatively few other undesirable structural attributes. Examples of undesirable structural attributes include, but are not limited to, restriction enzyme sites, eukaryotic sequence elements, vertebrate promoter components and transcription factor binding sites, response elements, E.coli sequence elements, mRNA secondary structure. Thus, optimized nucleic acid products may have increased levels of expression resulting from increased codon usage frequency and a reduced risk of inappropriate transcription behavior resulting from a reduced number of undesired transcriptional regulatory sequences.

An isolated and optimized nucleic acid molecule can have a codon composition that differs from a corresponding wild-type nucleic acid sequence by more than 30%, 35%, 40% or more, or 45% or more, e.g., 50%, 55%, 60% or more, of the codons. Exemplary codons for use in the invention are those codons that are used more frequently in a particular organism for the same amino acid than at least one other codon, and, in some embodiments, are also not low-use codons in that organism and are also not low-use codons in the cloned or screened organism for expression of the nucleic acid molecule. Also, codons for certain amino acids (i.e., those with three or more codons) can include two or more codons that are used more frequently than other (non-preferred) codons. The presence of codons in the nucleic acid molecule that are used more frequently in one organism than in another organism results in expression in cells of the organism at a level that is higher than the expression of the wild-type or parent nucleic acid sequence in those cells when the nucleic acid molecule using those codons that are used more frequently is introduced into those cells.

In some embodiments of the invention, the different codons are those codons that are used more frequently in mammals, while in still other embodiments, the different codons are those codons that are used more frequently in plants. Preferred codons for different organisms are known in the art, see, for example, http:// www.kazusa.or.jp./codon/. A particular type of mammal, such as a human, may have a different set of preferred codons than another type of mammal. Similarly, a particular type of plant may have a different set of preferred codons than another type of plant. In one embodiment of the invention, a majority of the different codons are codons that are preferred in the intended host cell. Codons preferred for organisms including mammals (e.g., humans) and plants are known in the art (e.g., Wada et al, Nucl. acids Res.,18:2367 (1990); Murray et al, Nucl. acids Res.,17:477 (1989)).

Examples

Reference example 1-Synthesis of α -aminonitrile (Compound 1):

the flask was filled with sodium bisulfite (71.4mmol) and 17mL of water. A14 mL Tetrahydrofuran (THF) solution containing an aldehyde (69.3 mmol) was added dropwise to the above flask at a rate such that the internal temperature was maintained at 60 ℃ or lower. The resulting suspension was stirred at room temperature for 40 minutes and ammonium hydroxide solution (4.85mL) was added over a period of 2 minutes. The resulting solution was magnetically stirred while being heated in an oil bath at 60 ℃ for 1 hour, and then left overnight at room temperature. The solution was cooled in an ice/brine bath until the measured internal temperature was below 5 ℃. 14mL of an aqueous solution containing sodium cyanide (71.4mmol) was added to the above solution over a period of 30 minutes. The resulting mixture was stirred at about 10 ℃ for 20 minutes, at 30 ℃ for 2 hours, and at room temperature for 18 hours. The reaction mixture was extracted into three 200mL portions of diethyl ether and the combined extracts were dried over anhydrous sodium sulfate. The mixture was filtered and the solution was cooled in an ice bath for 20 minutes, hydrogen chloride gas was added to the stirred solution until precipitation ceased, and the suspension was stirred for 1 hour. The solid was isolated by filtration and rinsed with three 50mL portions of diethyl ether. The material was dried in vacuo to yield 6.4g (47.5mmol) of a white solid (69%). The program is modified from: freifelder and Hasbrough, "Synthesis of Primary 1,2-Diamines by Hydrogenation of alpha-Aminonitriles," Journal of the American Chemical Society,82(3):696-698 (1960).

Reference example 2 Synthesis of 2-oxo-2-phenylacetaldoxime (Compound 2)

The flask was filled with potassium tert-butoxide (58mmol) and 63mL of tert-butanol. The mixture was stirred until a solution formed and 35mL of tert-butanol solution containing the appropriate benzophenone (50mmol) was added dropwise over a period of 15 minutes. The reaction mixture was stirred for 1 hour and pure isoamyl nitrite (75mmol) was added over 5 minutes. The completion of the reaction mixture was monitored and diluted with 100mL of heptane. The resulting solid (38mmol) was collected by suction filtration and dried under vacuum until constant weight. The program is modified from: hagedorn et al, chem. Ber.,98:193 (1965).

Reference example 3 Synthesis of pyrazine derivative (Compound 3)

The 3-neck flask was equipped with a thermometer, septum and argon line. Aminonitrile (47.5mmol), dried pyridine (190mL) and oxime (61.75mmol) were added thereto. The mixture was stirred thoroughly for 15 minutes and titanium tetrachloride (bis-pyridine) complex (94.9 mmol) was added in 5 portions over 35 minutes, ensuring that the internal temperature remained below 40 ℃. After the addition was complete, the reaction mixture was stirred at room temperature overnight. The reaction mixture was slowly added in small portions to a solution of sodium bicarbonate (174mL of water containing 21.75 g). The resulting mixture was stirred thoroughly for 15 minutes and 80g of celite was added. The suspension was stirred for 30 minutes and filtered through a Buchner funnel. The filtrate was transferred to a separatory funnel and the filter cake was suspended in 400mL of methanol. The mixture was stirred for 30 minutes and filtered again. This process was repeated four times. The methanol filtrates were combined and concentrated and the residue was dissolved in 200mL of ethyl acetate (EtOAc). The solution was added to a separatory funnel containing the original filtrate and the mixture was further extracted with three 100mL portions of EtOAc. The combined extracts were washed with two 100mL portions of saturated sodium carbonate and two 100mL portions of salt solution. The organic solvent was evaporated to give crude pyrazine oxide as a brown oil. The material was dissolved in 3mL of methanol and 89mL of Dichloromethane (DCM) was added. To the solution was added zinc powder (80.7 mmol), and the mixture was cooled in an ice bath until the internal temperature reached 15 ℃. The mixture was treated with glacial acetic acid (3 mL) and heated in an oil bath for 40 minutes to an internal temperature of 30 ℃. The reaction mixture was cooled to room temperature and filtered through a pad of celite. The filter cake was rinsed with DCM and the combined filtrates were washed with saturated aqueous sodium bicarbonate. The crude product was purified by chromatography on silica gel using a heptane/EtOAc gradient. This gave 2.9 g (29%) of pyrazine as a brown solid. The program is modified from: kishi et al, "The structure compliance of The light-emitting facility of The biomedical jellyfish," Tetrahedron letters, 13(27):2747 (1972).

Reference example 4 Synthesis of coelenterazine

The method A comprises the following steps: (the following compounds can be synthesized by method A: Compounds PBI-3840, PBI-3886, PBI-3857, PBI-3887, PBI-3913, PBI-3894, PBI-3896, PBI-3897, PBI-3841 and PBI-3842)

The flask was filled with pyrazine (8.25mmol), pyruvic acid (14.0mmol), camphorsulfonic acid (0.8 mmol) and anhydrous 2-methyl THF (150 mL). The flask was fitted with a condenser and soxhlet extractor with 4 angstrom molecular sieve and the reaction mixture was heated in an oil bath at 110 ℃ for 18 hours. The molecular sieves were replaced with fresh and reflux continued for 24 hours. The reaction mixture was filtered and concentrated, and the residue was dissolved in EtOAc (200 mL). The solution was washed with three 25mL portions of saturated sodium bicarbonate solution, 100mL of 0.1M sodium acetate buffer, pH 5, and 100mL of saline solution. The solution was dried over magnesium sulfate, filtered and concentrated to give 2.3g (6.2 mmol, 75%) of the crude enamine/acid. The material was dissolved in anhydrous THF (30mL) and the solution was cooled in an ice/water bath for 10 minutes. To this was added carbodiimide (9.0mmol) and pure diisopropylethylamine (14.9 mmol). After 10 minutes the cold water bath was removed and the reaction mixture was stirred at room temperature for 3 hours. To the reaction mixture was added 50mL of 0.1M sodium acetate buffer, pH 5, and the mixture was stirred thoroughly for 10 minutes. The biphasic mixture was extracted with three 100mL portions of EtOAc and the combined extracts were washed with a salt solution. The organic solvent was concentrated and the residue was purified by chromatography on silica gel using a DCM/methanol gradient. This gave 336mg (0.94mmol, 16%) of anhydrocoelenterazine as a red solid. The material was suspended in 10mL of methanol and the mixture was cooled in an ice bath. To this was added sodium borohydride (100mg, 2.6mmol) in three portions over a period of 1 hour. The reaction mixture was stirred for a further 30 minutes and pure glacial acetic acid was added dropwise until a pH of 5 was reached. The solution was concentrated and the residue was triturated with 15mL of water. The solid was isolated by suction filtration and dried under vacuum for several hours to give 318 mg (94%) of crude coelenterazine as a yellow solid. The program is modified from: kakoi and Inoue, chem.Lett. 11(3):299-300 (1980).

The method B comprises the following steps: (the following compounds can be synthesized by method B: Compounds PBI-3882, PBI-3932, PBI-3881)

The flask was filled with glyoxal (2.2mmol), aminopyrazine (1.1mmol), ethanol (20 mL), 12N hydrochloric acid (0.6mL), and water (1 mL). The reaction mixture was heated at reflux for 24 hours and concentrated. The residue was purified by column chromatography on silica gel using a DCM/methanol gradient. This gave 100mg (0.25mmol, 23%) of coelenterazine product as a black solid. The program is modified from: inoue et al "liquid biologics. II. isolation from Watasenia sciences and synthesis of 2- (p-hydroxyphenyl) -6- (p-hydroxyphenyl) -3, 7-dihydroimidazoi [1,2-a ] pyrazin-3-one" chem.Lett.,4(2):141-4 (1975).

The method C comprises the following steps: synthesis of novel coelenterazine (the following compounds can be synthesized by method C: PBI-3939, PBI-3945, PBI-3889, PBI-4002)

The compound 4- (5-amino-6-benzylpyrazin-2-yl) phenol can be prepared according to the methods described previously (Kishi et al, Tetrahedron Lett.,13:2747 (1972); Mosrin et al, Organic Letters,11:3406 (2009); Kakoi, chem. pharm. Bull.,50:301 (2002)).

And (3) synthesizing 2-amino-3-benzyl-5-phenylpyrazine. A round-bottom flask was charged with 5g (33.5 mmol) of 2-isonitroacetophenone, 6.7g (36.8mmol) of 2-amino-3-phenylpropionitrile hydrochloride, and 100mL of anhydrous pyridine. The mixture was cooled to-20 ℃ and 4.6mL (40.0 mmol) of TiCl were added dropwise ₄. The reaction was held at-20 ℃ for 30 minutes and heated to 80 ℃ for 2.5 hours. The solvent was evaporated and the residue was dissolved in 1L DCM. With saturated NaHCO₃And brine to wash the solution. All volatiles were evaporated and the residue was redissolved in ethanol (400 mL). Raney nickel (2.0g, aqueous suspension) was added and the reaction stirred under 1 atmosphere of hydrogen for 5 days. The mixture was passed through celite and the volatiles were removed. The residue was chromatographed on silica gel (heptane/DCM) to give 2.5g (29%) of 2-amino-3-benzyl-5-phenylpyrazine.

Synthesis of 2-amino-3-phenylpropanenitrile hydrochloride. The round bottom flask was filled with 65g (0.624mol) of sodium bisulfite and 150mL of water. 150mL of THF containing 75g (0.624mol) of phenylacetaldehyde were added dropwise. After stirring for 20 minutes, 37mL of 14M ammonium hydroxide were added in one portion and the mixture was heated to 60 ℃ for 60 minutes. After cooling to 0 ℃, the mixture was diluted with 150mL of water, and 100mL of water containing sodium cyanide (27.5g, 0.560mol) was added dropwise while maintaining the internal temperature below 10 ℃. After addition, the mixture was heated to 30 ℃ for 2 hours and extracted with diethyl ether. After drying over sodium sulfate, all volatiles were evaporated and the residue was dissolved in 3.5L of diethyl ether and treated with 400mL of 3.3M ethanolic HCl. The resulting precipitate was filtered and dried in vacuo to give 55g (60%) of the product.

Synthesis of 3- (furan-2-yl) -2-oxopropanoic acid. To a 100mL flask were added 3- (furan-2-yl) -2-oxopropanoate (940mg) and 23mL cold 6N NaOH. The insoluble mixture was stirred in a 90 ℃ bath for 5 minutes until dissolved. Cold 1N HCl was added until the solution was acidic (about 120 mL). The solution was extracted with 2X 50mL EtOAc. The combined organic layers were washed with 40mL of salt solution and Na₂SO₄And (5) drying. The solution was evaporated to give 540mg of a brown solid. The solid was further purified by reverse phase High Performance Liquid Chromatography (HPLC) gradient from 97% trifluoroacetic acid (TFA) to Acetonitrile (ACN).

Synthesis of ethyl 3- (furan-2-yl) -2-oxopropanoate. To a 500mL flask containing isomer (E/Z) -2-carboxamido-3- (furan-2-yl) acrylic acid ethyl ester (5.0g) was added 220mL of a frozen solution of 50/50 ethanol/water containing 1.4M (5%) HCl. After 5 h, the reaction was partitioned between 200mL EtOAc and 30mL brine. The aqueous layer was extracted with 2X 50mL EtOAc. The combined organic layers were washed with 1X 50mL of water and 1X 50mL of brine and washed over Na₂SO₄And (4) drying. The organic layer was coevaporated with 26g of celite and eluted with a gradient from heptane to EtOAc over 80g of gold silica. The appropriate combined fractions were evaporated to give 2.1 g.

Synthesis of (E/Z) -2-formylethyl-3- (furan-2-yl) acrylic acid Ethyl ester to a 500mL flask was added 50mL of diethyl ether, Cu₂O (320mg) and furan aldehyde (5.2 mL). The flask was cooled in an ice bath and ethyl 2-isocyanoacetate (5.3mL) was added. After 1.5 hours, (5g) potassium tert-butoxide was added to the reaction. After 4 hours, the heterogeneous reaction was filtered. 60mL of 30% citric acid and 20mL of EtOAc were added and stirred for 10 minutes. With 50mLEtOAc extracting the aqueous layer. The combined organic layers were dried over anhydrous sodium sulfate. The EtOAc layer was co-evaporated with 24g of celite and a gradient eluted from heptane to EtOAc over 80g of gold silica. The yellow slurry was used without further purification.

2-oxo-3- (thiophen-2-yl) propionic acid to a 250mL flask were added (E/Z) -5- (thiophen-2-ylmethylene) imidazolidine-2, 4-dione (5.0g) and 100mL of cold 6N NaOH. The mixture was heated to 100 ℃ for 1 hour. Concentrated HCl was added to the cooled solution until acidic (pH 1). The mixture was extracted with 8X 50mL diethyl ether. The combined ether layers were washed with 50mL brine over Na₂SO₄Dried and evaporated to give 3.36g of a solid. The sample was further purified by recrystallization using α, α, α -trifluorotoluene to give 1.63 g.

Synthesis of (E/Z) -5- (thien-2-ylmethylene) imidazolidine-2, 4-dione. Hydantoin (9.8g) and thiophene-2-carbaldehyde (10g) were added to a 250mL flask. To this mixture was added piperidine (9.6mL) dropwise. The mixture was heated to 100 ℃ for 1 hour and then poured into 300mL of 1N HCl. The solid was filtered, washed with water and dried in vacuo to give 4.9g of a solid.

Step 1-Add the appropriate phenylpyrazin-2-amine (100 mg), the appropriate pyruvic acid (2 equiv.), DCM (1mL) and 1,1, 1-trifluoroethanol (1mL) to a microwave vial (10mL) and heat stir at 80 ℃ for 30 min. The reaction was co-adsorbed on 2g of celite and the solvent was removed in vacuo. Celite was loaded onto 24g spherical silica gel and eluted with a gradient of heptane to ethyl acetate. The appropriate fractions were combined and evaporated.

Step 2-the material isolated in step 1 dissolved in THF (0.5mL) was frozen in an ice bath. Acetic anhydride (25. mu.L), dimethylaminopyridine (8.5mg) and triethylamine (25. mu.L) were added. After 2 hours, most of the THF was removed in vacuo. The product was precipitated with 30% aqueous citric acid (2 mL). The solid was washed with water (2mL) and then dissolved in 3mL of DCM. The DCM was washed with 1X 2mL of water followed by 1X 2mL of brine. The DCM layer was co-adsorbed on 2g of celite and the solvent was removed in vacuo. Celite was loaded onto 12g of spherical silica gel and eluted with a gradient of heptanes to DCM. The appropriate components were combined and evaporated.

Step 3-the material from step 2 dissolved in DCM (1mL) was frozen in an ice bath. To this solution were added methanol (0.5mL) and a solution of sodium borohydride in diglyme (325. mu.L, 0.5M). After 2 hours, acetic acid (10 μ L) was added and the solution was rapidly partitioned between 30% aqueous citric acid (1mL) and DCM (2 mL). The DCM layer was co-adsorbed on 1 g of celite and the solvent was removed in vacuo. Celite was loaded onto 4g spherical silica gel and eluted with a gradient of DCM to EtOAc. The appropriate fractions were combined and evaporated.

Step 4 (only when R ═ OAc) — the material in step 3 was dissolved in THF (200 μ L) and frozen in an ice bath. 1 equivalent of 1.35M potassium methoxide in THF was added to the solution. After 30 min, the reaction was partitioned between DCM (1mL) and 30% citric acid (1 mL). The DCM layer was co-adsorbed on 0.5g celite and the solvent was removed in vacuo. Celite was loaded onto 4g spherical silica gel and eluted with a DMC to EtOAc gradient. The appropriate fractions were combined and evaporated.

The method D comprises the following steps: (the following compounds can be synthesized by method D: the compounds PBI-3899, PBI-3900, PBI-3925, PBI-3933, PBI-3946) -in general, aminopyrazine is condensed with 2 equivalents of a 2-keto acid in the presence of a palladium catalyst under an atmosphere of hydrogen. Purification and subsequent activation of the resulting alpha-amino acid with respect to intramolecular condensation yields the corresponding imidazopyrazinone.

Example 5 Synthesis of 8-benzyl-6- (4-hydroxyphenyl) -2-propylimidazo [1, 2-. alpha. ] pyrazin-3 (7H) -one

2- ((3-benzyl-5- (4-hydroxyphenyl) pyrazin-2-yl) amino) pentanoic acid. 4- (5-amino-6-benzylpyrazin-2-yl) phenol (100mg, 0.36mmol) was mixed with ethanol (20mL) containing 2-oxopentanoic acid (84mg,0.72 mmol)And (4) mixing. Pd/C (10% palladium on activated carbon, 40mg) was added and the reaction mixture was heated to 65 ℃. By N₂The gas vented off the air and a hydrogen balloon was applied to the reaction flask. The reaction was stirred continuously for 4 hours. After cooling down, filtration was carried out and the resulting solution was purified by flash chromatography (eluting solvent: 50% EtOAc in heptane) to yield the product as a yellow powder (70mg, 52%).¹H NMR(300MHz,CD₂Cl₂,δ): 8.31(s,1H),7.82(d,2H,J＝9.0Hz),7.31(m,5H),6.92(d,2H,J＝ 9.0Hz),5.34(s,2H),4.20(m,1H),1.10(m,2H),0.98(m,2H),0.87(t, 3H)；MS(ESI)m/z 378.3(M+1)。

8-benzyl-6- (4-hydroxyphenyl) -2-propylimidazo [1,2-a]Pyrazin-3 (7H) -one. 2- ((3-benzyl-5- (4-hydroxyphenyl) pyrazin-2-yl) amino) pentanoic acid (49mg, 0.13mmol) was dissolved in DCM (10 mL). Pyridine (0.5mL) was added followed by N, N' -dicyclohexylcarbodiimide (54 mg, 0.26 mmol). The reaction mixture was stirred slowly at room temperature for 1 hour. The solvent was evaporated and the residue was purified by flash chromatography (eluting solvent: EtOAc to DCM to 10% methanol in DCM) to give the product as a yellow powder (40mg, 86%). ¹H NMR(300MHz, CD₃OD,δ):7.35(m,8H),6.88(d,J＝9.0Hz,2H),4.40(s,2H),2.81(t,J ＝7.5Hz,2H),1.81(m,2H),1.02(t,J＝7.5Hz,3H)；MS(ESI)m/z 359.0。

Example 6 Synthesis of 8-benzyl-2-butyl-6- (4-hydroxyphenyl) imidazo [1,2-a ] pyrazin-3 (7H) -one

2- ((3-benzyl-5- (4-hydroxyphenyl) pyrazin-2-yl) amino) hexanoic acid. 4- (5-amino-6-benzylpyrazin-2-yl) phenol (200mg, 0.72mmol) was mixed with ethanol (20mL) containing 2-ketohexanoic acid sodium salt (220mg, 1.44 mmol). Pd/C (10% palladium on activated carbon, 100mg) was added with a few drops of acetic acid and the reaction mixture was heated to 65 ℃. By N₂The gas vented and a hydrogen balloon was applied to the reaction flask. The reaction was stirred continuously for 4 hours. After cooling down, the resulting solution was filtered and purified by flash chromatography (eluting solvent: heptane with 50% EtOAc) to give the product as a yellow powder (130mg, 46%). MS (ESI): M/z 392.2(M + 1).

8-benzyl-2-butyl-6- (4-hydroxyphenyl) imidazo [1,2-a]Pyrazin-3 (7H) -one. 2- ((3-benzyl-5- (4-hydroxyphenyl) pyrazin-2-yl) amino) hexanoic acid (130mg, 0.33mmol) was dissolved in DCM (10 mL). Pyridine (0.5mL) was added followed by N, N' -dicyclohexylcarbodiimide (137mg, 0.67 mmol). The reaction mixture was stirred slowly at room temperature for 1 hour. The solvent was evaporated and the residue was purified by flash chromatography (eluting solvent: EtOAc to DCM to 10% methanol in DCM) to give the product as a yellow powder (110mg, 89%). ¹H NMR (300MHz,CD₃OD,δ):7.30(m,8H),6.88(d,2H),4.40(s,2H),2.84(t, 2H),1.77(m,2H),1.51(m,2H),0.89(m,3H)；MS(ESI)m/z 374.3(M+ 1)。

Example 7 Synthesis of 8-benzyl-2-ethyl-6-phenylimidazo [1,2-a ] pyrazin-3 (7H) -one (PBI-3925)

2- ((3-benzyl-5-phenylpyrazin-2-yl) amino) butyric acid. 3-benzyl-5-phenylpyrazin-2-amine (200mg, 0.77mmol) was mixed with ethanol (20 mL) containing 2-oxobutanoic acid (157mg,1.54 mmol). Pd/C (10% palladium in activated carbon, 100mg) was added and the reaction mixture was heated to 65 ℃. By N₂The gas was vented and a hydrogen balloon was applied to the reaction flask. The reaction was stirred continuously for 4 hours. After cooling down, filtration and purification of the resulting solution by flash chromatography (eluting solvent: 50% EtOAc in heptane) gave the product as a yellow powder (90mg, 34%).¹H NMR(300MHz,CD₂Cl₂,δ):7.72(s,1H),7.32-7.48(m, 10H),4.46(s,2H),4.20(m,2H),2.25(q,2H),0.99(t,3H)；MS(ESI): m/z 348.3(M+1)。

2- ((3-benzyl-5-phenylpyrazin-2-yl) amino) butyric acid was dissolved in DCM (10 mL). Pyridine (0.5mL) was added followed by N, N' -dicyclohexylcarbodiimide (137mg, 0.67 mmol). The reaction mixture was stirred slowly at room temperature for 1 hour. The solvent was evaporated and the residue was purified by flash chromatography (eluting solvent: EtOAc to DCM to 10% methanol in DCM) to give the product as a yellow powder (110mg, 89%).¹H NMR(300MHz,CD₃OD,δ): 7.26(m,3H),6.84-7.07(m,8H),4.03(s,2H),2.47(q,J＝9.0Hz,2H), 0.96(t,J＝9.0Hz,3H)；MS(ESI):m/z 330.2(M+1)。

Example 8 Synthesis of 8-benzyl-6-phenyl-2- (3,3, 3-trifluoropropyl) imidazo [1,2-a ] pyrazin-3 (7H) -one

5,5, 5-trifluoro-2-oxopentanoic acid. Ethyl 4,4, 4-trifluorobutyrate (1g, 5.88mmol) and diethyl oxalate (3.87g, 26.5mmol) were dissolved in ethanol. Sodium ethoxide (21% in ethanol, 2.09g) was added to the solution and the reaction mixture was stirred for 0.5 h. The solvent was distilled and the residue was extracted with EtOAc/water. The organic layer was collected and dried over sodium sulfate. After filtration, the solvent was removed to give a clear liquid. MS (ESI) M/z 269.1 (M-1). The liquid was then dissolved in 3N HCl (20mL) and the reaction mixture was refluxed for 4 hours. After cooling down, the reaction mixture was extracted with EtOAc. The organic layer was collected and dried over sodium sulfate. After filtration, the solvent was removed and the residue was used directly in the next step. MS (ESI) M/z 169.7 (M-1).

5,5, 5-trifluoro-2- ((3-benzyl-5-phenylpyrazin-2-yl) amino) butanoic acid. 3-benzyl-5-phenylpyrazin-2-amine (240mg, 0.92mmol) was mixed with ethanol (20mL) containing 5,5, 5-trifluoro-2-oxopentanoic acid (150 mg, 0.88 mmol). Pd/C (10% palladium in activated carbon, 100mg) was added and the reaction mixture was heated to 65 ℃. By N₂The gas was vented and a hydrogen balloon was applied to the reaction flask. The reaction was stirred continuously for 4 hours. After cooling down, filtration and purification of the resulting solution by flash chromatography (eluting solvent: heptane with 50% EtOAc) gave the product as a yellow powder (200mg, 54%). ¹H NMR(300MHz,CD₂Cl₂, δ):11.45(s,1H),10.20(s,1H),7.94(s,1H),7.34(m,10H),5.34(s,2H), 3.96-4.23(m,2H),3.02-3.28(m,2H)；FNMR:-76.3；MS(ESI):m/z 416.1(M+1)。

Coelenterazine (R)₁＝H，R₂＝-CH₂CH₂CF₃). 5,5, 5-trifluoro-2- ((3-benzyl-5-phenylpyrazin-2-yl) amino) butyric acid (100mg, 0.24mmol) was dissolved in DCM (10 mL). Pyridine (0.5mL) was added followed by N, N' -dicyclohexylcarbodiimide (100mg, 0.48 mmol). The reaction mixture was stirred slowly at room temperature for 1 hour.The solvent was evaporated and the residue purified by flash chromatography (eluting solvent: EtOAc to DCM containing 10% methanol) to give the product as a yellow powder (80mg, 87%).¹H NMR(300MHz,CD₂Cl₂,δ): 7.36(m,11H),3.43(s,2H),1.60-1.92(m,4H)；FNMR:67.4(t,J＝18Hz)； MS(ESI):m/z 398.2(M+1)。

Example 9 Synthesis of 8-benzyl-2- (furan-2-ylmethyl) -6-phenylimidazo [1,2-a ] pyrazin-3 (7H) -one (PBI-3939)

8-benzyl-2- (furan-2-ylmethyl) -6-phenylimidazo [1,2-a]Pyrazin-3 (7H) -one: the synthesis was performed by method C using 3- (furan-2-yl) -2-oxopropanoic acid and 3-benzyl-5-phenylpyrazin-2-amine as starting materials.¹H NMR (300MHz, dmso) δ 8.88(s,1H),8.02(d, J ═ 7.9,2H), 7.61-7.38 (m,6H), 7.37-7.14 (m,3H),6.38(s,1H),6.26(d, J ═ 3.2,1H),4.64(s,3H),4.40(s, 3H); for C₂₄H₂₀N₃O₂ ⁺The calculated accurate mass m/z +382.16, found m/z +382.

Example 10 Synthesis of 8-benzyl-6-phenyl-2- (thien-2-ylmethyl) imidazo [1,2-a ] pyrazin-3 (7H) -one (PBI-3889)

8-benzyl-6-phenyl-2- (thien-2-ylmethyl) imidazo [1,2-a ]Pyrazin-3 (7H) -one: the synthesis was performed by method C using 2-oxo-3- (thiophen-2-yl) propionic acid and 3-benzyl-5-phenylpyrazin-2-amine as starting materials.¹H NMR (300MHz, dmso) δ 8.85(s,1H),7.99(d, J ═ 6.8,2H), 7.63-7.02 (m,10H),6.94(dd, J ═ 3.5,5.1,1H),4.62(s,2H), 4.58(s,2H),2.69 (impurities); for C₂₄H₂₀N₃OS⁺Calculated accurate mass m/z + 398.13, measured m/z + 398.

Example 11 Synthesis of 8-cyclopropyl-2- (4-hydroxybenzyl) -6-phenylimidazo [1,2-a ] pyrazin-3 (7H) -one (PBI-3897)

8-cyclopropyl-2- (4-hydroxybenzyl) -6-phenylimidazo [1,2-a]Pyrazin-3 (7H) -one: synthesis was performed using 3-cyclopropyl-5-phenylpyrazin-2-amine and 3- (4-hydroxyphenyl) -2-oxopropanoic acid as starting materials using method a. For C₂₂H₁₈N₃O₂ ^-The calculated accurate mass m/z-356.14, the measured mass m/z-356.

Example 12 Synthesis of 8-benzyl-2-methyl-6-phenylimidazo [1,2-a ] pyrazin-3 (7H) -one (PBI-3932)

8-benzyl-2-methyl-6-phenylimidazo [1,2-a]Pyrazin-3 (7H) -one: synthesized using 1, 1-dimethoxypropan-2-one and 3-benzyl-5-phenylpyrazin-2-amine as starting materials using method B. For C₂₀H₁₈N₃O⁺The calculated accurate mass m/z +316.14, the measured mass m/z +316.

Example 13 Synthesis of 2- (4-hydroxybenzyl) -8-methyl-6-phenylimidazo [1,2-a ] pyrazin-3 (7H) -one (PBI-3896)

2- (4-hydroxybenzyl) -8-methyl-6-phenylimidazo [1,2-a]Pyrazin-3 (7H) -one: the synthesis was carried out using method a using 3-methyl-5-phenylpyrazin-2-amine and 3- (4-hydroxyphenyl) -2-oxopropanoic acid as starting materials.¹H NMR(300MHz,dmso)δ8.84(s,1H),8.00(d,J ＝7.6,2H),7.47(dd,J＝8.6,16.2,3H),7.17(d,J＝7.3,2H),6.69(d,J＝ 8.4,2H),6.26(s,4H),4.17(s,2H),2.86(s,3H),2.48(s,1H)。

Example 14 Synthesis of 8-benzyl-2- (4-hydroxybenzyl) -6-phenylimidazo [1,2-a ] pyrazin-3 (7H) -one (PBI-3840)

8-benzyl-2- (4-hydroxybenzyl) -6-phenylimidazo [1,2-a]Pyrazin-3 (7H) -one: the synthesis was carried out using method A using 3- (4-hydroxyphenyl) -2-oxopropanoic acid and 3-benzyl-5-phenylpyrazin-2-amine as starting materials. For C₂₆H₂₂N₃O₂ ⁺The calculated accurate mass m/z +408.17, the measured mass m/z +408.

Example 15 Synthesis of protected coelenterazine (stabilized) (PBI-4377)

To a mixture of PBI-3939, potassium carbonate (1.1 eq) and potassium iodide (1.1 eq) in Dimethylformamide (DMF) was added 1 eq of chloromethyl pivalate at room temperature under an argon atmosphere. The progress of the reaction was monitored by thin layer chromatography and, after completion, the reaction mixture was cooled in an ice bath for a few minutes and then the same volume of water as the reaction volume was added. The resulting mixture is extracted with a suitable organic solvent (e.g., EtOAc), and the extract is concentrated to give the crude product. The material was further purified by chromatography on silica gel.

Example 16-8-benzyl-2- ((1-methyl-1H-imidazol-2-yl) methyl) -6-phenylimidazo [1,2-a ] pyrazin-3 (7H) -one (PBI-4525), synthesis of 8-benzyl-6- (4-hydroxyphenyl) -2- ((1-methyl-1H-imidazol-2-yl) methyl) -6-phenylimidazo [1,2-a ] pyrazin-3 (7H) -one (PBI-4540) and 2- ((1H-imidazol-2-yl) methyl) -8-benzyl-6-phenylimidazo [1,2-a ] pyrazin-3 (7H) -one (PBI-4541).

To a flask containing 10mmol of 2- methylimidazole derivative 1 or 2 was added 20mL of anhydrous THF under an argon atmosphere, and the solution was cooled to about-78 ℃ in a dry ice/acetone bath. To this cooled mixture was added dropwise a solution of 9.3mmol of n-butyllithium (2.46M in hexane) over a period of several minutesAnd (4) liquid. The resulting solution was stirred at about-78 ℃ for 30 minutes and 6.7mmol of compound 3 was added via syringe. The reaction mixture was stirred for 3 hours and quenched by the addition of 20mL of saturated ammonium chloride solution and 20mL of saturated sodium bicarbonate solution. The cold bath was removed and after warming to room temperature, the mixture was extracted with 3 × 100mL of EtOAc. Drying (MgSO)₄) The combined extracts were concentrated in vacuo and crude compound 4 or 5 was purified by column chromatography using silica gel (EtOAc/heptane).

Microwave vials were filled with 100mg (1 equivalent) of compound 6 and 7 and 2 equivalents of compound 8 or 9. To this mixture was added 4.5mL of ethanol and 0.25mL of concentrated HCl. The reaction mixture was heated in a microwave at 100 ℃ for 1.5 hours. The resulting reaction mixture was added to 50mL of EtOAc and then washed with 20mL of saturated solution of catalase and 20mL of brine. The organic layer was concentrated in vacuo and the residue was purified by column chromatography using silica gel (methanol/dichloromethane) to give compound 10-12.

Example 17 stability and self-luminescence characterization of novel coelenterazine

The stability and self-luminescence characteristics of the novel coelenterazine PBI-3939, PBI-3889, PBI-3945, PBI-4002 or PBI-3896 were determined. Higher stability and lower self-luminescence are attractive technical features in the substrate/reagent.

To determine stability, 20. mu.M of the novel coelenterazine PBI-3939, PBI-3889, PBI-3945, PBI-4002 or PBI-3896, 30. mu.M of native coelenterazine or 22. mu.M of known coelenterazine-h or known coelenterazine-hh were placed in a medium containing 50mM CDTA, 150mM KCl, 50mM DTT, 35mM thiourea, 1%

NP-9(v/v) and 0.1%

DF 204 in reporter buffer. The replicated samples were incubated at room temperature (i.e., 22-24 ℃) for various lengths of time and then transferred to-70 ℃. After all samples were collected and frozen, they were thawed and mixed with phenol red-free + 0.1%

40 μ L of DMEM containing OgLuc variant IV in 10 μ L of bacterial cell lysate. Luminescence of the samples was read 5 minutes after IV addition.

“T₉₀"represents the amount of time taken for the luminescent signal to decay by 10% (i.e., a 10% reduction in activity) at room temperature, i.e., 22 ℃. The slope of the linear fit of the data plotted from ln RLU versus time determines the rate of luminescence signal decay ("T ₉₀") which is calculated by the following equation: t ═ ln (A/A)₀) Div (-k), where A ═ intensity at time t, A₀ Time 0, and k the rate of decay. As shown in Table 1, T of known coelenterazine-h and-hh, novel coelenterazine PBI-3939, PBI-3889, PBI-3945, PBI-4002 and PBI-3896₉₀Values higher than native coelenterazine indicate that these coelenterazine are more stable compounds than native coelenterazine.

To determine the self-luminescence profile, HEK293 cells were cultured overnight at 15,000 cells per well in DMEM + 10% FBS + pyruvate. CO-independent media removal and use in addition of 10% FBS ₂20 μ M of each of the novel coelenterazines shown in FIG. 2, namely PBI-3939, PBI-3889, PBI-3945, PBI-4002, PBI-3841, PBI-3897, PBI-3896, PBI-3925, PBI-3894, PBI-3932 and PBI-3840, native coelenterazine and known coelenterazine, coelenterazine-h and coelenterazine-hh, were diluted in the medium of (A). Immediately after the addition of the substrate

Luminescence was measured on a Luminometer (1 sec/well). The background luminescence was 154. + -. 15 RLU. Table 1 shows the self-luminescence characteristics normalized for native coelenterazine ("self-luminescence (normalized for coelenterazine)"). Coelenterazine-h has more self luminescence than native coelenterazine, but all other coelenterazines examined have less self luminescence.

Table 1: stability experiments and self-luminescence characterization of IV Using different coelenterazines

Example 18 toxicity of novel coelenterazine

Toxicity of the novel coelenterazine was studied in HEK293 cells. HEK293 cells were grown overnight at 15,000/well in DMEM + 10% FBS + pyruvate. Medium was removed and used in CO-independent cultures containing 10% FBS₂Replaced with the diluted novel coelenterazine compound (or DMSO control). After 24 hours of addition of the compound, CELLTITER-

Assay reagents (Promega Corp.) measure cell viability. And is arranged at

Luminescence was measured on a Luminometer (1 sec/well). Table 2 shows the toxicity of novel coelenterazine, known coelenterazine-h and coelenterazine-hh, and novel coelenterazine PBI-3939, PBI-3889, PBI-3841, PBI-3897, PBI-3945, PBI-4002, and PBI-3840 in HEK293 cells. The novel coelenterazine has at least the same toxicity as coelenterazine-hh, except for PBI-3840. Some of the novel coelenterazines have the same toxicity as native coelenterazines and coelenterazines-h.

Table 2: according to CELLTITER-

Toxicity of different coelenterazines in HEK293 cells

EXAMPLE 19 Km of PBI-3939

To determine Km of PBI-3939, use was made according to the manufacturer's instructions

Protein Purification System through

Fusion purified OgLuc variant L27V (described in example 26) and contained no phenol red and 0.1%

Diluted in DMEM. mu.L of assay buffer (100mM MES pH 6, 35mM thiourea, 0.5%

NP-9(v/v), 1mM CDTA, 2mM DTT and 150mM KCl) with varying amounts of PBI-3939 were added to 50. mu.L of diluted enzyme (about 20pM final enzyme concentration) and luminescence was measured at 22 ℃ for 3 minutes. As shown by the data in FIG. 3, PBI-3939 has a Km of about 10 μ M.

Example 20 characterization of the Compounds PBI-4525, PBI-4540 and PBI-4541

Compounds PBI-4525, PBI-4540 and PBI-4541 were screened for the ability to detect luminescence. For analysis, 20. mu.M of each compound was added to assay buffer (100mM MES pH 6, 35mM thiourea, 0.5%

NP-9(v/v), 1mM CDTA, 2mM DTT and 150mM KCl), adjusted to pH 7 with 100mM HEPES pH 7 to construct assay reagents. Then, the mixture is added to a mixture containing no phenol red and 0.1 percent

The assay reagents were mixed with 36pM purified L27V02 enzyme (described in example 25B). As a control, an assay buffer containing 20. mu.M PBI-3939 or PBI-4528 was used. In the measurement ofLuminescence was measured as described previously 3 minutes after the reagent was added to the enzyme mixture. Table 3 shows that the compounds PBI-4525, PBI-4540 and PBI-4541 can be used to detect luminescence from luciferase utilising coelenterazine.

TABLE 3

Example 21 OgLuc Pattern sequence

Families of enzymes comprising different luciferase species can be identified by having a common three-dimensional structure and defined catalytic activity. Because enzyme families share evolutionary history with other enzyme families, they will also show similarities in their three-dimensional structures. Through different modes of structural and functional analysis, the present inventors have identified OgLuc as representative of decapod luciferases, having a three-dimensional structure significantly similar to Fatty Acid Binding Protein (FABP), indicating a commonality of the evolutionary history. Thus, a decapod luciferase can be defined as having a characteristic three-dimensional structure similar to FABP and using coelenterazine as a substrate to catalyze the emission of luminescence. Other luciferases, e.g., firefly luciferase, Renilla luciferase, bacterial luciferase and similar luciferases, have significantly different three-dimensional structures, suggesting that they belong to different enzyme families and do not share evolutionary history. Dinoflagellate (Dinoflagellate) luciferases have a three-dimensional structure that shows some similarity to FABPs, suggesting a shared evolutionary history, but do not utilize coelenterazine as a substrate, and thus do not belong to the same family of enzymes as decapod luciferases.

Because amino acid sequences are not as conserved as three-dimensional structures, defining enzyme families based solely on sequence comparisons can be difficult. For example, even though FABPs have a characteristic barrel-shaped three-dimensional shape, comparisons of their amino acid sequences often show very low levels of sequence identity. Nevertheless, sequence identity can be used to indicate commonality of three-dimensional structures. If the amino acid sequences of two proteins can be aligned to show > 30% sequence identity, preferably > 40% sequence identity, and most preferably > 50% sequence identity, the two proteins will have similar three-dimensional structures (Chothia and Lesk, EMBO J.5(4): 823-. Thus, a protein is a decapod luciferase if, based on its amino acid sequence alignment with the sequence of OgLuc, the sequence identity is > 30%, preferably > 40%, and most preferably > 50%, and the protein can utilize coelenterazine as a substrate to catalyze the emission of luminescence.

Some portions of the amino acid sequences in the enzyme family exhibit a higher amount of conservation (i.e., a higher level of sequence homology) due to structural limitations necessary to maintain the characteristic three-dimensional structure of the enzyme family. Thus, these conserved regions can be used as additional evidence of a common three-dimensional structure shared between two proteins. Conserved sequence patterns, also known as tags, motifs or blots, can be generated by artificial or computer methods known in the art. Patterns can be found in public databases such as PROSITE (http:// expay. org/place; signist et al, Nucleic Acids res.38 (supplement 1): D161-D166 (2010)).

For example, patterns of conserved amino acids can be found when examining large numbers of known FABPs. PROSITE (release 20.67, 5/10/2010) contains the FABP pattern (accession number PS00214, constructed 4/1990, data updates to 4/2006). The FABP pattern spans 18 amino acid positions and is defined as follows:

[GSAIVK]-{FE}-[FYW]-x-[LIVMF]-x-x-{K}-x-[NHG]-[FY]-[DE] -x-[LIVMFY]-[LIVM]-{N}-{G}-[LIVMAKR](SEQ ID NO: 329)(VI)，

wherein:

standard IUPAC single letter codons for amino acids were used.

The symbol "x" is used to accept any amino acid position.

By listing amino acids between brackets "[ ]" to indicate alternative amino acids at a site (e.g., [ ALT ] represents the likelihood of Ala, Leu, or Thr at that position).

The presence of a particular amino acid at a site is indicated by curly brackets "{ }" (e.g., { AM } represents any amino acid at a position other than Ala and Met).

Each sequence position (or element in the pattern) is separated from its neighbors by "-".

Each sequence position is called a "mode position", e.g., [ GSAIVK ] will be considered mode position 1 of formula (VI), { FE } is considered mode position 2 of formula (VI), etc.

Although conserved sequences are due to a common underlying three-dimensional structure, some changes to the sequence pattern that do not disrupt the three-dimensional structure may be allowed. For example, for some members of the FABP family, differences were found at four sites in the PROSITE pattern. These additional members of the FABP family include the 5 proteins listed in PROSITE as false negative coincidences, i.e., FABP protein family members not included in the FABP pattern (UniProt database accession numbers FBP12_ HUMAN, FABP1_ FASGI, FABP2_ fahe, FABPL _ SCHBI, RET5_ BOVIN) and one protein known to have FABP folds (protein database accession number 2a 02). Although OgLuc shares a closely similar three-dimensional structure with FABP, the sequence patterns of the native and variant amino acid sequences are also slightly different, with differences from the PROSITE pattern at 5 positions. In various embodiments, the pattern in OgLuc begins at a position corresponding to position 8 of SEQ ID NO. 1. The pattern of amino acid substitutions, deletions or insertions is calculated as a difference.

In combination with sequence information from these additional FABPs and OgLuc variants, improved sequence patterns can be deduced:

[GSAIVK]-{FE}-[FYW]-x-[LIVMFSYQ]-x-x-{K}-x-[NHGK]-x-[ DE]-x-[LIVMFY]-[LIVMWF]-x-{G}-[LIVMAKRG](SEQ ID NO: 330)(VII).

the sequence information used to derive this pattern is shown in table 4. Column 1 identifies the pattern position (listed from N-terminus to C-terminus; pattern length is 18 amino acids) and column 6 identifies the corresponding sequence position in OgLuc (numbered according to SEQ ID NO: 1). Column 2 shows the PROSITE FABP schema (equation (VI)) elements for each schema location. Column 3 lists amino acids represented by non-PROSITE FABP patterns present in FABP family members. Column 4 lists the amino acids present in OgLuc (SEQ ID NO:1) or OgLuc variants that are not represented by the PROSITE pattern. Column 5 lists the modified schema ("OgLuc schema") constructed by fusing schema information from columns 2, 3, and 4 (equation (VII)). Column 7 lists the amino acids in the OgLuc corresponding to the PROSITE FABP pattern position. Column 8 lists the amino acids found in the dinoflagellate luciferase sequences (8 different species) at positions corresponding to the improved pattern (GenBank accession numbers 2021262A, AAA68491, AAC36472, AAV35379, AAV35380, AAL40676, AAL40677, AAV35378, AAV35377, AAV35381, and protein database accession number 1 VPR).

The modified pattern (formula (VII)) serves as an indicator (i.e., a blot) of the three-dimensional protein structure shared between FABP and OgLuc. However, strict agreement with this pattern is not required to indicate commonality of three-dimensional structures. From the example given here, a common three-dimensional structure may exist even if there are as many as 5 changes in the pattern. Also, for example, even though the dinoflagellate luciferase has a similar three-dimensional structure to FABP and OgLuc, it has 4 differences from the modified pattern.

Thus, even if the protein can be recognized as a decapod luciferase based on sequence similarity and luminescence using coelenterazine, it can be further recognized by having an improved sequence pattern at the same time. In particular, if based on an alignment of its amino acid sequence with SEQ ID No. 1 or a variant thereof, the sequence identity is > 30%, preferably > 40%, and most preferably > 50%, and the protein can use coelenterazine as a substrate to catalyze the emission of light, and the amino acid sequence starting at the position corresponding to position 8 of SEQ ID No. 1 is the following:

[GSAIVK]-{FE}-[FYW]-x-[LIVMFSYQ]-x-x-{K}-x-[NHGK]-x-[ DE]-x-[LIVMFY]-[LIVMWF]-x-{G}-[LIVMAKRG](SEQ ID NO: 330)(VII)，

having no more than 5 differences, or more preferably no more than 4, 3, 2 or 1 differences, or more preferably no differences, wherein a difference occurs in a position corresponding to mode position 1, 2, 3, 5, 8, 10, 12, 14, 15, 17 or 18 of formula (VII) according to table 4, then the protein is a decapod luciferase. The differences may also include gaps or insertions between the mode positions of table 4.

Table 4: protein sequence patterns

Example 22 Generation of OgLuc variants

Details of the experiment

Unless otherwise indicated, additional variants of the starting OgLuc variant sequence with Random substitutions were generated using the error-prone, mutagenic PCR-based system GeneMorph II Random Mutagenesis Kit (Stratagene; Daughery, PNAS USA,97(5):2029(2000)) according to the manufacturer's instructions and NNK site saturation (Zheng et al, Nucleic Acids Research, 32: e115 (2004)).

Additional variants with specific mutations starting OgLuc variants were generated using the oligonucleotide-based Site-Directed Mutagenesis Kit QuikChange Site-Directed Mutagenesis Kit (Stratagene; Kunkel, PNAS USA,82(2):488(1985)) according to the manufacturer's instructions.

The resulting variants were used in pF1K for expression based on the T7 promoter

Vectors (Promega Corp.) were constructed in the environment. Alternatively, the resulting variant was constructed in the context of a pF4Ag vector (a commercially available form of pF4A (Promega Corp.)), which contained modifications including those with or without a C-terminus

T7 of the E.coli ribosome binding site and the CMV promoter (Ohana et al, Protein Expression and Purification,68:110-120(2009)) to produce a fusion Protein. For example, to obtain the C1+ A4E variant, NNK saturation mutagenesis experiments were performed in a pF1K vector background. The C1+ A4E library was generated in a pF4Ag vector background without HT 7. The QC27, QC27-9a, and IVY libraries were generated in a pF4Ag vector background with C-terminal HT 7. IV-based variants were generated in the context of pF4Ag vector without HT 7. The resulting vector is used to transform KRX E.coli using techniques known in the art.

The resulting OgLuc variants are named with respect to the amino acid substitutions identified in the variants and/or with respect to the e.coli clone contained in the variants, e.g., figure 6A shows that e.coli clone 16C5 has the substitution Q20R, among others.

Details of screening

The resulting library was expressed in e.coli and initially screened using an automated system for changes in relative specificity or variants of OgLuc with increased light output (i.e., increased luminescence, increased brightness, or increased light emission) as compared to the corresponding starting OgLuc variant. The automated initial screening performed was as follows: individual clones from the generated library were used to inoculate minimal medium in 96-well plates and grown at 37 ℃ for 17 to 20 hours ("M1 culture"). M1 was diluted 1:20 in fresh minimal medium and incubated at 37 ℃ for 17-20 hours ("M2 incubation"). M2 culture was diluted 1:20 into induction medium and grown at 25 ℃ for 17-20 hours with walk-through induction, i.e., auto-induction (Schagat et al, "KRX Autoinduction Protocol: A Convenient Method for Protein expression," Promega Notes,98:16-18 (2008)). When novel coelenterazine PBI-3841, PBI-3842, PBI-3857, PBI-3880, PBI-3881, PBI-3886, PBI-3887, PBI-3897, PBI-3896 or PBI-3894 is used as a substrate in the initial screening, the induction medium contains rhamnose and glucose. When natural coelenterazine, known coelenterazine-h or novel coelenterazine PBI-3840, PBI-3889, PBI-3899 or PBI-3900 is used as substrate in the initial screening, the induction medium is free of rhamnose or glucose. The use of different induction media was determined from the luminescence generated between C1+ A4E and the novel coelenterazine, i.e. rhamnose and glucose containing induction media was used with the novel coelenterazine generating less luminescence with C1+ A4E compared to other novel coelenterazines with C1+ A4E.

mu.L of the induced cells were lysed with 60. mu.L lysis buffer containing 300mM HEPES pH 8.0, 300mM thiourea, 0.3X passive lysis buffer ("PLB"; Promega Corp. catalog No. E194A), 0.3mg/mL lysozyme and 0.003U/. mu.L RQ1 DNase, and with 150mM KCl, 1mM CDTA, 10mM DTT, 0.5%

NP-9(v/v) in 50. mu.L assay buffer to measure luminescence and utilize 20. mu.M of native, known or novel coelenterazine as substrate. Luminescence measurements were performed for each variant 3 minutes after reagent addition and relative luminescence unit values were normalized to the average of 8 control wells of the corresponding starting OgLuc variant per plate. In that

The assay is completed on an automated system.

The OgLuc variants of interest are sequenced using standard sequencing techniques known in the art to identify any added amino acid substitutions in each of the variants. Secondary screening was performed on variant clones of interest using a non-automated (manual) system. The manual screening was performed as follows: variant clones were grown in triplicate in 96-well plates and allowed to express, the assay was performed as described for the automated assay, but the assay buffer was added manually using a multichannel pipettor. For each variant, luminescence was measured, averaged and normalized with respect to the corresponding starting OgLuc variant. By using

Luminescence measurements were performed with an F500 luminometer.

Determining changes in relative specificity

The relative substrate specificity is determined by dividing the luminescence of the luciferase in the presence of the test coelenterazine substrate by the luminescence of the luciferase in the presence of the reference coelenterazine substrate. For example, relative specificity is determined by dividing the luminescence of the luciferase using the novel coelenterazine of the present invention by the luminescence of the luciferase using a different coelenterazine (e.g., a native or known coelenterazine, or a different novel coelenterazine of the present invention). The test coelenterazine substrate and the compared reference coelenterazine substrate are considered a comparison substrate pair for determining relative substrate properties.

The change in relative substrate specificity is determined by dividing the relative substrate specificity of a test luciferase using the comparison substrate pair by the relative substrate specificity of a reference luciferase using the same comparison substrate pair. For example, the change in relative specificity is determined by dividing the relative substrate specificity of the test luciferase of the novel coelenterazine of the invention as compared to a different coelenterazine (e.g., a native or known coelenterazine or a different coelenterazine of the invention) by the relative substrate specificity of a reference luciferase of the same novel coelenterazine of the invention as compared to the same different coelenterazine used to test the luciferase.

Luminescence with one novel coelenterazine was compared to luminescence with a different novel coelenterazine. Luminescence using one native or known coelenterazine is compared to luminescence using another native or known coelenterazine. Luminescence using a native or known coelenterazine was compared to luminescence using the novel coelenterazine.

An increase in luminescence (RLU) of the OgLuc variant and a decrease or no change in luminescence of the reference coelenterazine as compared to the corresponding starting OgLuc template for the novel coelenterazine indicates a relatively specific change. A reduction in luminescence of the OgLuc variants of both the novel and reference coelenterazine compared to the corresponding starting OgLuc, while a more reduced luminescence of the OgLuc variant with the novel coelenterazine, also indicates a change in relative specificity. An increase in luminescence of the OgLuc variant compared to the corresponding starting OgLuc for the novel and reference coelenterazine indicates an increase in activity/stability/expression. If the luminescence of the OgLuc variant is increased for both the novel and reference coelenterazine, and the increase in luminescence for the novel coelenterazine is higher, an increase in the relative specificity and an improvement in activity/stability/expression of the OgLuc variant is indicated.

C1+ A4E variants

C1+ A4E (SEQ ID NOS: 2 and 3), previously described in U.S. application No. 12/773,002 (U.S. published application No. 2010/0281552), was used as the basic starting sequence (i.e., parental sequence) for generating additional synthetic OgLuc variants. C1+ A4E has the following amino acid substitutions relative to SEQ ID NO: 1: A4E, Q11R, a33K, V44I, a54F, P115E, Q124K, Y138I, and N166R. The luminescence of C1+ A4E containing bacterial lysate was measured as previously described and compared to the luminescence using native and known coelenterazine as substrate (FIGS. 5A-G) using the novel coelenterazine described in examples 1-14 (see FIG. 4 for example) as substrate. FIG. 5A shows luminescence using native coelenterazine ("coelenterazine"), known PBI-3880 and novel coelenterazine PBI-3842, PBI-3857, PBI-3881, PBI-3882, PBI-3886 and PBI-3887 as substrate C1+ A4E. Luminescence measurements using known and novel coelenterazine the light field was normalized using the luminescence of C1+ A4E of native coelenterazine and reduced by fold compared to native coelenterazine (fig. 5B). FIGS. 5C-E show luminescence from C1+ A4E using native coelenterazine and novel coelenterazine PBI-3945, PBI-3894 and PBI-4002, respectively. FIG. 5F shows the luminescence of C1+ A4E using native coelenterazine and novel coelenterazine PBI-3840, PBI-3897, PBI-3889, PBI-3899, and PBI-3900. FIG. 5G shows the luminescence of C1+ A4E using native coelenterazine, known coelenterazine PBI-3912 and new coelenterazine PBI-3913, PBI-3925, PBI-3939, PBI-3933, PBI-3932, PBI-3946, PBI-3841 and PBI-3896. The data indicate that the C1+ A4E variant can utilize each of the novel coelenterazines as substrates.

Unless otherwise indicated, the resulting C1+ A4E variants have at least the amino acid substitutions identified in C1+ A4E. A library of 4400 variant clones of C1+ A4E (library 1) was generated by random mutagenesis as previously described and screened as previously described for improvements in relative specificity changes and/or activity changes (e.g., brightness). Variants were initially screened using native coelenterazine, known coelenterazine-h (known as PBI-3880) and novel coelenterazine PBI-3840, PBI-3841, PBI-3842, PBI-3857, PBI-3881, PBI-3886, PBI-3887, PBI-3889, PBI-3897 and PBI-3900 as substrates. In addition, one half of the variants were screened using novel coelenterazine PBI-3896 and PBI-3894 as substrates. Plates containing variants with known mutations of interest identified from screening for previous novel compounds were selected. Variants that show improvements (relative specificity changes or activity changes) in the primary screening assay with respect to the novel coelenterazine or coelenterazines tested are isolated, sequenced and screened in a secondary screening.

In the secondary artificial screening, the known coelenterazine PBI-3912, coelenterazine-h, coelenterazine-hh, 2-methyl coelenterazine and coelenterazine v are utilized; and novel coelenterazine PBI-3840, PBI-3897, PBI-3889, PBI-3899, PBI-3900, PBI-3925, PBI-3944, PBI-3932, PBI-3945, PBI-3913, and PBI-3896 as substrates. FIGS. 6A-D summarize the mean luminescence for variants normalized to C1+ A4E ("clones"). Figures 6A-D summarize substitutions ("amino acid sequences") in these variants with at least one of the following added amino acid substitutions: a14V, G15R, Q18L, Q20R, L22I, E23K, L27V, L27M, K33N, T39I, E49K, F54S, F54I, D55G, I56V, V58I, V58L, I59T, S66T, G67S, F68S, L72Q, M75K, I76N, F77T, F77C, K89E, I90V, I90T, L92H, H93R, M106K, Y109F, P113T, I117F, T126R, V127A, L136M, D139G, P145L, S148T, C164S, or a 169V.

As shown in fig. 6A-C, amino acid substitutions at positions 54, 92 and 109 are of interest because substitutions at these positions provide higher light output or improved relative specificity, i.e., specificity that is far from native coelenterazine and close to at least one novel coelenterazine (as shown in fig. 6A-C). The amino acid substitution F54I in clone 29H7 provided higher light output with some of the native coelenterazine and the novel coelenterazine. The amino acid substitution Q18L in clone 40H11, the amino acid substitution L92H in clone 04a12, and the amino acid substitution Y109F in clone 43F9 provided improved relative specificity.

Table 5 lists C1+ A4E variants generated as previously described with additional amino acid substitutions ("amino acid changes") at positions 77, 92 or 109. These variants were analyzed for increased light output as previously described, i.e., variants that were at least 1.3 × brighter than C1+ A4E were screened using native coelenterazine, known coelenterazine-hh and novel coelenterazine PBI-3939, PBI-3894, PBI-3896, PBI-3897, PBI-3932 or PBI-3925 as substrates. The following additional substitutions resulted in variants at least 1.3 × brighter than C1+ A4E: L92G, L92Q, L92S, L92A, L92M, L92H, L92Y, F77W, F77Y, F77S, F77T, F77V, F77A, F77G, F77C, F77D, F77M and Y109F. As shown in Table 5, the L92H, F77W, and F77A substitutions made the most significant improvements with PBI-3897, PBI-3896, and PBI-3932.

Table 5: saturation of the 77 th, 92 th and 109 th sites

Additional variants of C1+ A4E (group A) were generated by site-directed mutagenesis as previously described to have additional substitutions relative to SEQ ID NO:1 in at least one of the following amino acid positions: 18. 20, 54, 59, 72, 77, 89, 92, 109, 113, 127, 136, or 164. These amino acid positions were selected because, based on primary and secondary screening of library 1, substitutions at these positions had increased overall light output compared to C1+ A4E using at least one of the following as a substrate: novel coelenterazine PBI-3841, PBI-3896, PBI-3897, PBI-3894, PBI-3925 or PBI-3932 or known coelenterazine 2-methyl coelenterazine or PBI-3912. Figure 7 lists the variants ("clones") contained in each variant and the additional amino acid substitutions. Variant clones were assayed in triplicate as described for secondary manual screening and normalized to C1+ A4E as previously described. FIGS. 8A-B and 9 show normalized mean luminescence for the variants listed in FIG. 7 using different coelenterazine as substrates. Figures 8A-B and 9 show a greater increase in luminescence of the novel compounds listed compared to C1+ A4E or no change or decrease in luminescence of known coelenterazine compared to C1+ A4E. Clone QC27, which had additional amino acid substitutions Q18L, F54I, L92H, and Y109F, had a 561.32-fold increase in luminescence with PBI-3896, a 392.98-fold increase with PBI-3894, and an 283.85-fold increase with PBI-3896 compared to C1+ A4E. This data shows that Q18L, L92H, and Y109F can be combined with each other and have additional substitutions to produce variants with improved relative specificity.

Other permutations of interest identified from library 1 were combined to generate additional variants (panel B) (fig. 10). Additional amino acid substitutions are made relative to SEQ ID NO 1 in at least one of the following amino acid positions: 18. 20, 54, 71, 77, 90, 92, 109 or 127. These substitutions show improvements using at least one of the following novel coelenterazines as a substrate: PBI-3841, PBI-3896, PBI-3897, PBI-3894, PBI-3925 or PBI-3932. These variants were assayed as described for group A variants using native coelenterazine, known coelenterazine-hh and novel coelenterazine PBI-3939, PBI-3945, PBI-3840, PBI-3932, PBI-3925, PBI-3894 and PBI-3896. Variant clones were assayed in triplicate as described for secondary manual screening and normalized to C1+ A4E as previously described. Figure 11 shows the normalized mean luminescence of the variants listed in figure 10 using different coelenterazine as substrate. Figure 11 shows a substantial increase in luminescence of the novel coelenterazine listed compared to C1+ A4E or no change or decrease in luminescence of native and known coelenterazine compared to C1+ A4E.

Additional variants were generated using additional amino acid substitutions I90V and/or Y109F (group C) and compared to the variants generated from group a or group B (see figure 12). Clones containing variants with the I90V substitution ("I90V"), the Y109F substitution ("Y109F") or both substitutions ("LE 2") were compared to clones QC #27, QC # 2E 7, QC # 2F 4 and QC #1 a11 using native coelenterazine, known coelenterazine-hh and novel coelenterazine PBI-3939, PBI-3945, PBI-3889, PBI-3840, PBI-3925, PBI-3932, PBI-3894, PBI-3896 and PBI-3897 as substrates using assays as described for group a recombinants (fig. 12). Variant clones were assayed in triplicate as described for the secondary manual screen and normalized to C1+ A4E as previously described (fig. 12). Fig. 12 shows a substantial increase in luminescence of the novel coelenterazine listed compared to C1+ A4E and no change or decrease in luminescence of native or known coelenterazine compared to C1+ A4E. Figure 12 shows that I90V provides higher light output for some of the native coelenterazine and novel substrates.

QC27 variants

Variant QC27(SEQ ID NOS: 4 and 5) from A with additional amino acid substitutions Q18L, F54I, L92H and Y109F was cloned into pF4A modified vectors as previously described to construct C-terminal HT7(Promega Corp.) fusion proteins ("QC 27-HT 7") (SEQ ID NOS: 44 and 45). 4400 variants of QC27-HT7 (library 2) were generated by random mutagenesis as previously described and primary screening for increased relative specific changes was performed as previously described using native coelenterazine and novel coelenterazine PBI-3896 and PBI-3897 as substrates. Variant clones were selected in a secondary manual screen using native coelenterazine, known coelenterazine-hh, and novel coelenterazine PBI-3897, PBI-3896, and PBI-3894 as substrates for sequencing and assays.

FIG. 13 lists additional amino acid substitutions identified in these variants ("samples") ("sequences"), and luminescence normalized to the corresponding starting QC27-HT7 using native coelenterazine, known coelenterazine-hh, and novel coelenterazine PBI-3897, PBI-3896, and PBI-3894 as substrate variants in a secondary screen. The variant in figure 14, having at least one of the following additional amino acid substitutions: F1I, R11Q, L18I, L18Q, V21L, V21M, L22F, F31I, Q32H, V45E, L46Q, S47P, G48P, E49P, G51P, D55P, G67P, F68P, Q69P, L72P, E74P, M75P, I76P, H86P, I90P, H92P, T96P, V98P, I99P, V102P, M106P, F109P, L142P, V158P, T159P, L168P or G36170 (the G36170 is located in the lugc region between the oght variants and the G P).

The amino acid substitution F68Y in variant 24B12, L72Q in variant 29C4, and M75K in variant 3H11 each provided higher light output for some of the native coelenterazine and novel substrates. The amino acid substitution V21L in variant 25a11 and H92R in variant 1B6 provided increased relative specificity. Both substitutions are cases where the luminescence signal is reduced using the novel coelenterazine as a substrate, but is more reduced using the natural and known coelenterazine as a substrate.

Additional QC27-HT7 variants were generated using site-directed mutagenesis as previously described to have specific amino acid substitutions (fig. 14). Additional substitutions were made at least one of the following amino acid positions relative to SEQ ID NO: 1: 21. 68, 72, 75, 76, 90, 92 and 158, since these positions show an increase in relative specificity change as shown in FIG. 14. FIG. 15 shows the luminescence normalized to the corresponding starting QC27-HT7 using native coelenterazine, known coelenterazine-hh, and the novel coelenterazines PBI-3897, PBI-3841, PBI-3896, and PBI-3894 as substrate QC27-HT7 variants. As seen in figure 15, combining the three amino acid substitutions F68Y, L72Q and M75K with V158I, as exemplified in variant QC27#1, provided higher light output for each coelenterazine tested.

QC27-9a variants

The library was generated using variant QC27-9a from B (SEQ ID NOS: 6 and 7), i.e., a QC27-HT7 fusion protein with additional amino acid substitutions V21L, H29R, F68Y, L72Q, M75K, and V158I, as the starting sequence. 4400 variants of QC27-9a (library 3) were generated by random mutagenesis as previously described and screened for increased relative specificity changes using native coelenterazine and novel coelenterazine PBI-3841 and PBI-3897. Variant clones were selected in a secondary manual screen as previously described using native coelenterazine, known coelenterazine-hh, known coelenterazine-h and novel coelenterazine PBI-3841 and PBI-3897 as substrates for sequencing and assays. FIG. 16 lists additional substitutions identified in the variants ("samples") ("amino acid changes"), and mean luminescence for the corresponding starting QC27-9a using native coelenterazine, known coelenterazine-hh, known coelenterazine-h, and novel coelenterazine PBI-3841 and PBI-3897 as substrate variants in a secondary screen. An increase in relative specificity represents a decrease in luminescence with the novel, natural and known coelenterazine variants compared to the starting template, but a more decreased luminescence with the natural and known coelenterazine. For example, variant 30D12, which has the amino acid substitution L22F, has about a three-fold loss of activity with novel coelenterazine PBI-3841 and PBI-3897. However, with native coelenterazine, known coelenterazine-h and known coelenterazine-hh, the luminescence of variant 30D12 was reduced by a factor of 10 or more.

FIG. 17 shows luminescence of C1+ A4E, QC27-HT7, and QC27-9a compared to humanized Renilla luciferase (referred to herein as "hRL") (SEQ ID NOS: 30 and 31) using native coelenterazine, known coelenterazine-hh, and novel coelenterazine PBI-3841 and PBI-3897 as substrates. Although the reaction of QC27-9a with PBI-3897 was brighter than QC27-9a with PBI-3841 (see FIG. 17), the evolution trend, i.e., the increased intensity of light emission, was the highest with respect to PBI-3841 (Table 6). The combination of an increase in luminescence (440-fold) and a decrease in luminescence (800-fold) of native coelenterazine suggests a change in relative specificity (350,000-fold) using PBI-3841 QC27-9a compared to native coelenterazine.

Table 6: alteration of relative specificity of OgLuc variants for PBI-3897 and PBI-3841 as compared to native coelenterazine and coelenterazine-hh

IVY variants

IVY (SEQ ID NOS: 8 and 9), the C1+ A4E variant with additional amino acid substitutions F54I, I90V, and F77Y, was cloned into pF4A modified vectors as previously described to construct the C-terminal HT7 fusion protein ("IVY-HT 7"). The 4400 variants of IVY-HT7 (library 4) were generated by random mutagenesis and screened for increased light output (i.e., increased brightness) and increased relative specificity using native coelenterazine, known coelenterazine-hh and the novel coelenterazines PBI-3840, PBI-3889, PBI-3925, PBI-3932 and PBI-3945 as substrates. Variant clones were selected in three replicates in a secondary screen as previously described using native coelenterazine, known coelenterazine-hh and novel coelenterazine PBI-3889, PBI-3939, PBI-3945 and PBI-4002 as substrates, and sequenced and assayed. FIGS. 18 and 19 list additional substitutions identified in the variants ("samples") ("amino acid changes") and the mean luminescence for IVY-HT7 with native coelenterazine, known coelenterazine-hh and novel coelenterazine PBI-3889, PBI-3939, PBI-3945, and PBI-4002 as substrate variants in a secondary screen. Figure 18 lists those variants (group a) selected based on performance using PBI-3945 with at least one of the following amino acid substitutions: Q18H, D19N, Q20P, Q32P, K33N, V38I, V38F, K43N, I44F, E49G, I60V, Q69H, I76N, Y77N, Y94F, G95S, G95D, F110I, V119M, K124M, L149I, or R152S. Figure 19 lists those variants (group B) selected based on performance using PBI-3889 with at least one of the following amino acid substitutions: F6Y, Q18L, L27V, S28Y, Q32L, K33N, V36E, P40T, Q42H, N50K, G51R, H86L, N135D or I155T.

Additional IVY-HT7 with additional amino acid substitutions were generated using site-directed mutagenesis as previously described. FIG. 20 lists variants with at least one of the following additional amino acid positions relative to SEQ ID NO: 1: 19. 20, 27, 32, 38, 43, 49, 58, 77, 95, 110 and 149, which show specificity close to that of PBI-3945 and PBI-4002, as these substitutions were identified in the variant of figure 18. FIG. 21 shows the luminescence of the variants listed in FIG. 20 normalized to IVY-HT7 using native coelenterazine, known coelenterazine-h, known coelenterazine-hh and the novel coelenterazines PBI-3939, PBI-3945, PBI-4002, PBI-3932 and PBI-3840 as substrates. None of the variants showed an increase over IVY-HT7, but there are examples such as variant C5.19(SEQ ID NOS: 12 and 13) which showed about a 3-4log reduction in luminescence with native or known coelenterazine, but only a two-fold reduction in luminescence with PBI-3945 and PBI-4002. The variant C5.19 has further amino acid substitutions L27V, V38I and L149I.

FIG. 22 lists at least one of the following increased amino acid positions relative to SEQ ID NO: 1: 6. 18, 27, 28, 33, 34, 36, 40, 50, 51, 135 and 155, as these substitutions were identified in the variant of figure 19, which shows specificity close to that of PBI-3889 and PBI-3939. FIG. 23 shows the luminescence of the variants listed in FIG. 21 using native coelenterazine, known coelenterazine-hh and novel coelenterazine PBI-3939, PBI-3945, PBI-3889, PBI-4002, PBI-3932 and PBI-3840 as substrates and normalized with respect to IVY-HT 7. The luminescence of each variant was reduced compared to IVY-HT 7. Variant C1.3(SEQ ID NOS: 10 and 11) using PBI-3939 has about 2000-fold higher luminescence than using native or known coelenterazine. Variant C1.3 has additional amino acid substitutions F6Y, K33N, N135D and I155T.

The optimal IVY-HT7 variant with altered relative specificity compared to hRL and IVY-HT7 is C5.19, which has optimal luminescence with PBI-3945, and C1.3, which has optimal luminescence with PBI-3889. FIG. 24 shows luminescence using native coelenterazine, known coelenterazine-h, known coelenterazine-hh and novel coelenterazine PBI-3939 and PBI-3945hRL, IVY-HT7, C5.19 (C-terminal HT7 fusion) and C1.3 (C-terminal HT7 fusion).

E.IV variants

IV (SEQ ID NOS: 14 and 15), a C1+ A4E variant with additional amino acid substitutions F54I and I90V, was generated as previously described. To determine the brightest variant for use as a transcription reporter, luminescence provided by C1+ A4E (SEQ ID NOS: 2 and 3), IVY (SEQ ID NOS: 8 and 9) and IV (SEQ ID NOS: 14 and 15) was measured as previously described using native coelenterazine, known coelenterazine and the novel coelenterazines PBI-3939, PBI-3945, PBI-3889 and PBI-4002 as substrates. hRL was used as a control. As seen in fig. 25, IV is brighter than both C1+ A4E and IVY. The amino acid substitution F54I in IV provided higher light output for some of the native coelenterazine and the novel substrates. All three variants of coelenterazine tested were brighter than hRL.

Data from A, B and D (i.e., screening of libraries generated from C1+ A4E, IVY, and QC27 as starting sequences) were combined to determine those additional amino acid substitutions with multiple coelenterazine with increased light output (i.e., increased brightness). IV variants with additional substitutions having a two-fold to ten-fold reduced specificity for native coelenterazine were generated as previously described. As listed in figure 26, the IV variants ("clones") have additional amino acid substitutions ("sequences") of at least one of the following amino acid substitutions: F1I, E4K, Q18L, L27V, K33N, V38I, F68Y, M70V, L72Q, M75K or V102E.

Variant clones from 16 plates of all combinations of amino acid substitutions were subjected to primary screening and assayed using native coelenterazine, known coelenterazine-h, known coelenterazine-hh and novel coelenterazine PBI-3889 and PBI-3945 as substrates as previously described using automated robotic methods. Variants with improved luminescence were selected in three replicates using manual screening as previously described for sequencing and assay. Luminescence was measured using native coelenterazine, known coelenterazine-h, known coelenterazine-hh and the novel coelenterazines PBI-3889, PBI-3939, PBI-3945 and PBI-4002 as substrates. The corresponding starting sequences IV and hRL were used as controls.

Figure 26 lists variants, and additional amino acid substitutions identified in the variants. Figure 27 shows the mean luminescence of variants in the secondary screen with respect to IV normalization. Variant 8A3(SEQ ID NOS: 26 and 27), which has additional amino acid substitutions F1I, L27V and V38I, has increased relative specificity with the novel coelenterazine, but is less bright than IV. Variant 8F2(SEQ ID NOS: 46 and 47), which has the additional amino acid substitution L27V, provided increased relative specificity and brightness using 3 of the 4 novel coelenterazines used. Variant 9B8(SEQ ID NOS: 18 and 19), which has additional amino acid substitutions Q18L, F68Y, L72Q and M75K, is brighter than all substrates and also provides some relative specificity advantages over native coelenterazine. Variant 9F6(SEQ ID NOs: 20 and 21) with additional amino acid substitutions Q18L, L27V, V38I, F68Y, L72Q and M75K, showed similar increases as seen with 8F 2. Variant 15C1(SEQ ID NOs: 16 and 17) with additional amino acid substitutions E4K, K33N, F68Y, L72Q and M75K, which are brighter for all novel coelenterazine, but do not have any improved relative specificity benefits. In the case of IV, the amino acid substitution Q18L in variant 1D6 provided increased relative specificity, i.e., was far from native coelenterazine and close to the novel substrate. In summary, the amino acid substitution L27V provided relative specificity in the case of IV.

FIG. 28 shows the luminescence of 8A3, 9B8, 9F6 and 15C1 in a secondary screen using native coelenterazine, known coelenterazine-hh, known coelenterazine-h and novel coelenterazine PBI-3939, PBI-3945, PBI-3889 and PBI-4002 as substrates compared to IV and hRL. The brightness compared to IV using the native coelenterazine variant 8a3 had a 2log reduction. The brightness compared to IV using the native coelenterazine variant 9F6 had a 1log reduction. Variant 15C1 using PBI-3945 was the brightest, but had a short signal half-life (see example 27).

F.9B8 variant

The 9B8 variant from E was further modified to generate additional variants with increased light emission and/or increased relative specificity for PBI-3939. The amino acid substitution L72Q represents an advantageous amino acid substitution for increased light emission (i.e., brightness) because the substitutions were identified in variants 9B8, 9F6, and 15C1, all of which showed increased light emission. To determine whether additional amino acid substitutions at position 72 would provide similar increases in brightness, additional variants of 9B8 were generated as previously described by saturating position 72 with the alternative residue. Four replicates of E.coli lysate were prepared and analyzed for brightness as previously described using PBI-3939 as substrate, except that the assay buffer contained 10mM CDTA, 150mM KCl, 10mM DTT, 100mM HEPES, pH 7.0, 35mM thiourea and 0.5%

NP-9 (v/v). Table 7 lists 9B8 variants ("variants") with similar or increased luminescence compared to 9B8, i.e., fold increase, as indicated by the luminescence normalized for 9B8 ("RLU (normalized for 9B 8)"). The amino acid substitutions A, G, N, R and M at position 72 provided at least the same brightness benefit as amino acid Q, i.e., a 1-fold increase.

Table 7: a variant with similar luminescence compared to variant 9B 8.

Variants	RLU (standardized with respect to 9B 8)
		9B8+Q72A	1.1
9B8+Q72G	1
		9B8+Q72N	1
9B8+Q72R	1
		9B8+Q72M	1

Additional variants with increased relative specificity for the novel PBI-3939 were generated as previously described by saturating amino acids 18, 68, 72, 75 and 90 in variant 9B 8. Coli lysates were prepared and analyzed for brightness as previously described using native coelenterazine and novel PBI-3939 as substrates. Relative specificity was determined from the ratio of the luminescence with the variant of PBI-3939 to the luminescence with the variant of the novel coelenterazine, with respect to the corresponding luminescence normalization of 9B 8. Table 8 lists 9B8 variants ("variants") with at least a 1.1X fold increase in relative specificity for PBI-3939. The results indicate that at least one additional alteration at each site provides relative specificity for PBI-3939 as compared to native coelenterazine. The 9B8 variant with amino acid substitutions K, D, F, G, Y, W and H at position 18 had the highest fold increase in relative specificity.

Table 8: variants with increased relative specificity for PBI-3939

G.9B8+ K33N variants

An additional variant, 9B8opt + K33N (SEQ ID NOS: 42 and 43) was generated to investigate the benefits of the amino acid substitution K33N on brightness, relative specificity and thermostability. 9B8opt + K33N was examined in various applications and compared to 9B8opt (described in example 25A).

Coli lysates containing the variants 9B8opt or 9B8opt + K33N were prepared and analyzed as described previously, except that the assay buffer contained 0.1%

NP-9 (v/v). Luminescence generated from the lysate was measured using novel PBI-3939 and native coelenterazine as substrates. The relative specificity of the variants was calculated as described previously for PBI-3939 and native coelenterazine. 9B8opt + K33N ("K33N") had higher light output (RLU) than native coelenterazine and higher relative specificity for PBI-3939 compared to 9B8opt (FIG. 29), indicating that the K33N replacement provided higher light output and increased relative specificity.

A novel OgLuc library was constructed using 9B8opt + K33N as the starting template. By using

A Random library was constructed from the PCR Random Mutagenesis Kit (ClonTech; catalog # 630703). Condition 5 (as listed in the user manual) was used to generate additional variants, with an average mutation calculated by running sequence data from 83 randomly selected clones to 2.6 mutations per gene. This PCR library was cloned into a pF4 Ag-based non-fused vector background and sandwich background as previously described, i.e., Id-OgLuc-HT7 (described in example 45). pF4 Ag-based non-fusion vector background Designated as (NF). The variant in the background of sandwich vector was named (F). To clone the PCR product into both vectors, an amino acid, glycine, was attached to the variant sequence in pF4Ag, creating a new position 170 ("170G") in the OgLuc variant. This 170G is present in the sandwich construct, but in this case is considered to be part of the linker between OgLuc and HT 7. For each library, 4,400 E.coli clones were assayed as previously described except as follows. Lysis buffer contained 300mM MES pH 6.0 instead of HEPES, and 0.5%

NP-9(v/v), but no thiourea. The assay buffer contained 100mM MES pH 6.0 instead of HEPES, and 35mM thiourea. The assay volume was as follows: 10 μ L of cells, 40 μ L lysis buffer and 50 μ L assay buffer.

The PCR library in the non-fused background based on pF4Ag was screened for additional variants with increased luminescence compared to 9B8 opt + K33N +170G (SEQ ID NOS: 68 and 69). The selected variants were then assayed in HEK293 cells and NIH3T3 cells. For each cell type, 15,000 cells were seeded and grown overnight at 37 ℃. The following day, 10ng of pGL4.13(Promega Corp.) transfected cells as described in example 25 were used as transfection control, and 100ng of the OgLuc test DNA was used to transfect the cells. The medium was removed and the cells lysed with 100. mu.L of lysis buffer as described in example 25, except that the lysis buffer contained 100mM MES pH 6.0 instead of HEPES and utilized

Luminometer measures luminescence. For each sample, 10. mu.L of lysate was assayed using 50. mu.L of lysis buffer containing 20. mu.M PBI-3939. For transfection control, 50. mu.L of BRIGHT-GLO was used^TM Assay reagents 10. mu.L of lysate was assayed.

Table 9 shows the fold increase in luminescence of the variants in e.coli, HEK293 and NIH3T3 cells and the amino acid substitutions found in the variants. Variants 27A5(NF) (SEQ ID NOS: 70 and 71), 23D4(NF) (SEQ ID NOS: 72 and 73) and 24C2(NF) (SEQ ID NOS: 74 and 75) had at least a 1.3-fold increase in luminescence in E.coli cells and HEK293 cells.

Table 9: increase in luminescence generated by OgLuc variants compared to 9B8 opt + K33N +170G in E.coli cells, HEK293 cells and NIH3T3 cells

Based on the above data, other combinatorial variants were designed and generated in the context of the non-fused vector background based on pF4Ag without 170G (see table 10). Variants were analyzed in enterobacter coli cells, HEK293 cells, and NIH3T3 cells as described above and compared to 9B8 opt + K33N. Luminescence using variants of native coelenterazine was examined. Table 10 shows the luminescence of the variants in e.coli cells, HEK293 cells and NIH3T3 cells, and the amino acid substitutions found in the variants ("samples"). Variants are named by adding additional amino acid substitutions in the variant to the prefix "9B 8 opt + k33n. Table 11 shows the relative specificity of the different variants for PBI-3939 compared to native coelenterazine in e.coli cells, NIH3T3 cells and HEK293 cells. As shown in FIG. 10, the variant 9B8 opt + K33N + T39T + K43R + Y68D ("V2"; SEQ ID NO:92 and 93) had increased luminescence in E.coli and a slight increase in luminescence in NIH3T3 cells. Of the three cell types examined, the variant 9B8 opt + K33N + L27V + K43R + Y68D ("L27V, K43R, Y68D") had increased neutrality of luminescence (table 10) and relative specificity exceeding the 5X fold increase of 9B8 opt + K33N (table 11).

Table 10: increase in luminescence generated by OgLuc combinatorial variants in escherichia coli cells, NIH3T3 cells, and HEK293 cells compared to 9B8 opt + K33N

Table 11: alteration of the relative specificity of OgLuc combination variants for PBI-3939 compared to native coelenterazine in E.coli cells, NIH3T3 cells, and HEK293 cells

Generating from 9B8 opt + K33N a further OgLuc variant comprising at least one of the following further amino acid substitutions relative to SEQ ID NO: 1: L27V, T39T, K43R, Y68D or S66N (see "samples" in table 12 for amino acid substitutions in variants). Variants are named by adding additional amino acid substitutions in the variant after the prefix "9B 8 opt + k33n. These further variants and variants from the above were examined for brightness, relative specificity, signal stability and thermostability 9B8 opt + K33N + L27V + Y68D ("L27V, Y68D"), 9B8 opt + K33 8 + L27 8 + K43 8 + Y68 8 ("L27 8, K43 8, Y68 8"), 9B8 opt + K33 8 + L27 8 + K43 8 + S66 8 ("L27 8, K43 8, S66 8") and 9B8 opt + K33 8 + T39 8 + K43 8 + Y68 8 ("T39 8, K43 8, Y68 8"; also referred to as "V8"). The variants were compared to variants 9B8 opt ("9B 8") and 9B8 opt + K33N ("K33N").

Coli lysates containing the variants were prepared and analyzed as previously described. Luminescence generated from the lysate was measured using novel PBI-3939 and native coelenterazine as substrates. The luminescence of the variants was normalized with respect to the luminescence generated by 9B8 opt (table 12). The relative specificity of the variant for PBI-3939 and native coelenterazine was calculated by dividing the luminescence of the variant using PBI-3939 as a substrate by the luminescence of the variant using native coelenterazine as a substrate (table 12). The data indicate that the amino acid substitution L27V reduced specificity for native coelenterazine.

Table 12: increase in luminescence generated by the OgLuc variant compared to 9B8 and alteration of the specificity of the OgLuc variant for PBI-3939 compared to native coelenterazine in bacterial lysates

Sample (I)	Over a multiple of 9B8	Exceeds the multiple of coelenterazine
			9B8	1.0	7
K33N	1.1	21
			T39T，Y68D	0.9	12
T39T，L27V，K43R	1.2	149
			L27V，T39T，K43R，Y68D	1.8	110
T39T，K43R，Y68D	1.6	21
			L27V，T39T，K43R，S66N	1.3	114
L27V，K43R，Y68D	1.3	110
			L27V，Y68D	1.0	63
L27V，K43R，S66N	1.1	114

H.V2 variants

A set of additional variants were designed using V2 as template (9B 8opt with additional amino acid substitutions K33N + T39T + K43R + Y68D). Based on 1) the diversity known from the structure-based alignment of 28 fatty acid binding proteins (1VYF, 1FDQ, 2A0A, 1O8V, 1BWY, 2ANS, 1VIV, 1PMP, 1FTP, 2HNX, 1jj, 1CBR, 2CBS, 1LPJ, 1KQW, 2RCQ, 1EII, 1CRB, 1IFC, 2PYI, 2JU3, 1MVG, 2QO4, 1P6P, 2FT9, 1MDC, 1O1U, 1 EIO; see U.S. published application No. 2010/0281552), or 2) probes for the role of alternative residues at previously identified positions in substrate specificity the substitutions shown in table 13 were designed. The changes listed under "consensus sequences" in table 13 relate to residues identified in at least 50% of the aligned fatty acid binding proteins described above. The changes listed under "major minor" relate to residues identified in most of the above-mentioned fatty acid binding proteins but found below 50% of the aligned sequences. The changes listed under "other" relate to residues less frequent than the predominant minority residue at a given position in the aligned sequences. Finally, the changes listed under "specificity" relate to the positions presumably involved in determining the specificity of a variant for coelenterazine or a coelenterazine analog. For example, the designed specificity change at position 27 (leucine residue in the parent sequence, i.e., V2) is changed to another hydrophobic residue or to an amino acid representing a substitute chemistry (e.g., a hydrophobic residue comprising a loop, a residue comprising an uncharged polar side chain, or a residue comprising a charged side chain); and the designed specificity change at position 40 (proline in the parent sequence) was a choice for a different chemistry (e.g., other hydrophobic residues containing loops, residues containing uncharged polar side chains or residues containing charged side chains); note that glycine, glutamine, isoleucine and leucine were identified as aligned fatty acid binding proteins in this position).

Watch 13

Variants were constructed using standard site-directed mutagenesis protocols (see previous examples) and the resulting plasmids were transformed into E.coli for analysis. Cultures were grown according to standard walk-through induction in minimal medium as previously described. mu.L of lysis buffer (100mM MES pH 6.0, 0.3X PLB, 0.3mg/mL lysozyme, 0.003U/. mu.L RQ1 DNase I and 0.25% each after the other was added to 10. mu.L of cultured transformed E.coli cells

NP-9(v/v)) and equal volume (50. mu.L) of assay buffer (1mM CDTA, 150mM KCl, 2mM DTT, 20. mu.M PBI-3939 or native coelenterazine, 100mM MES pH 6.0, 35mM thiourea and 0.5%

NP-9 (v/v)). In that

Luminescence was measured on a 96 Microplate Luminometer (Promega Corp.).

Table 14 summarizes the different amino acid substitutions identified in the analysis. Data are presented for the parental clone (V2) with respect to the normalization of the luminescence measured for PBI-3939 and native coelenterazine. The relative change in specificity for PBI-3939 with respect to native coelenterazine is also shown.

TABLE 14

L27v variants

Using the OgLuc variant L27V as a starting template, i.e., the starting or parent sequence, additional variants were generated in which some of the amino acids in the L27V variant (table 15) were reverted to the amino acids found in the native OgLuc luciferase of SEQ ID NO: 1. Variants were constructed by site-directed mutagenesis as previously described. The variants were then screened for relative activity using native coelenterazine or PBI-3939 as previously described. 5 minutes after addition of substrate/assay reagent (as described in H) at

Luminescence was measured on F500 and normalized to the L27V variant starting template. SDS-PAGE analysis of the lysates indicated similar expression levels (data not shown).

Table 15 shows the relative activity using native coelenterazine or PBI-3939L 27V variants. Relative activity<1 indicates that reversion is detrimental compared to the residue at this site in the L27V variant. Relative activity>1 indicates that the recovery is favorable for activity compared to the residue at this site in the L27V variant. Some additional data for these mutants suggest the following: the thermal stability of 166K, 54F, 54A and L27V was examined. T of 166K, 54F and 54A _1/287, 74 and 33% at 60 ℃ respectively, indicating that these substitutions resulted in a reduction in thermal stability. The Km values for the same 4 variants were as follows: for native coelenterazine, L27V was 16 μ M, 54A was 23 μ M, 54F was 40 μ M, and 166K was 21 μ M; for PBI-3939, L27V was 18 μ M, 54A was 62 μ M, 54F was 163 μ M, and 166K was 23 μ M. This indicates a higher substrate affinity of L27V, especially with respect to the substitution at position 54.

Watch 15

Example 23 mutation analysis at position 166

A. To evaluate the effect of the different amino acids at position 166 on luciferase activity, the arginine (R) residue at position 166 was substituted for each of the other 19 amino acids in the context of the pF4Ag vector (i.e., in the context of the wild-type OgLuc sequence SEQ ID NO: 1) as previously described. These 166 nd variants were then expressed in E.coli as previously described.

To construct the lysate, 50. mu.L of 0.5X FASTBREAK^TMCell lysis reagent (Promega Corp.) was added to 950 μ l of induced culture and the mixture was incubated at 22 ℃ for 30 minutes. For analysis, 50 μ L of lysate was assayed in 50 μ L of assay reagent (as previously described in example 22H) using 100 μ M PBI-3939, 30 μ M native coelenterazine, or 22 μ M coelenterazine-H. Luminescence was measured as previously described (fig. 30A-C). FIGS. 30A-C show the relative specificity of the N166 mutants. Western blot analysis confirmed similar expression for all variants (data not shown).

B. Specific individual amino acid substitutions L27V, a33N, K43R, M75K, T39T, L72Q, and F68D were evaluated in the context of wild-type OgLuc or N166R. Individual amino acid substitutions were generated by site-directed mutagenesis as described previously, expressed in E.coli as described previously and luminescence measured using assay reagents with 22. mu.M native coelenterazine (as described previously in example 22H) (FIG. 30D). Western blot analysis confirmed similar expression for all variants (data not shown).

Example 24 deletion variants

Deletions were made to the L27V variant as follows:

TABLE 16

Deletion #	Deletion of generation
		27	Residues 1-27 and Val-1
52	Residues 1-52 and Val-1
		64	Residues 1-64 and Val-1
84	Residues 1-84 and Val-1
		19	Residues 65-83
23	Residues 65-87
		23A1	Residues 65-87+ G64D

The N-terminus of the OgLuc variant L27V is methionine, valine, and phenylalanine, i.e., MVF. For numbering purposes, phenylalanine is considered the first amino acid, and "Val-1" indicates that valine in "MVF" is deleted. Methionine of "MVF" is contained in these deletions. The L27 deletion variant was cloned into pF4Ag vector for expression in e.coli KRX cells as previously described. Induction and lysate preparation were performed as described, lysates were analyzed using assay reagents (previously described; 100. mu.M PBI-3939), and side-dose luminescence was performed as previously described (FIG. 31). The data demonstrate that smaller fragments of the OgLuc variant can also produce luminescence.

Example 25 codon optimization of OgLuc variants

A.IV and 9B8

IV and 9B8 OgLuc variants were used as templates for codon optimization. As understood by those skilled in the art, the purpose is two-sided: 1) removal of known transcription factor binding sites or other regulatory sequences that may be capable of interfering with the regulation or expression of the OgLuc variant, e.g., promoter modules, splice donor/acceptor sites, splice silencers, Kozak sequences and polyadenylation signals, and 2) alteration of DNA sequences (via silent mutations that do not alter protein sequences) to eliminate rare codons and to favor the codons most commonly used in cells of e.coli, humans, other mammals or other eukaryotic organisms (Wada et al, Nucleic Acids res., 18:2367 (1990)).

Two different optimized sequences of IV and 9B8, called opt (a.k.a. optA) and optB, were designed for each variant. An optimized sequence, opt/optA, was designed for each variant by identifying the two best, i.e. most common, human codons for each site (see table 17) and then randomly selecting one of the two for incorporation at each site. For the opt B form, previously, an optimized form of codon usage, i.e., opt/opt A, was used as the starting template, and each codon was replaced by the other of the two best human codons identified for this codon optimization strategy. As an example, the leucine amino acid at position 3 in the IV sequence or the 9B8 sequence is encoded by the codon TTG. TTG is not one of the two most common codons for leucine in human cells and thus this codon is changed to a replacement, the more frequently used codon for leucine, CTC (opt/optA) or ctg (optb). This same process is repeated for all leucines in the sequence and the CTC codons may terminate at optB and the CTG at optA due to the random nature of the process. Due to the optimization of this two-codon usage approach, the largest codons of the sequences opt/opt A and opt B differ.

Table 17: codons used in codon optimization

Each of the 4 sequences (IV opt, IV optB; 9B8 opt, 9B8 optB) was analyzed for the presence of transcription factor binding sites or other regulatory sequences as described above (genomic Software, Germany) and these unwanted sequences were destroyed by silent nucleotide changes. In some cases, where other non-rare codons for human and e.coli are present, the transcription factor binding site or other regulatory element is removed by changing to one of these codons, even if they are not selection #1 or selection #2 (see table 18). In these cases, where removal of the transcription factor binding site or other regulatory element would involve the introduction of a rare codon, the transcription binding site (or other regulatory element) is not typically altered.

Table 18: additional codons for removal of transcription factor binding sites and other regulatory elements

Amino acids	Selection #	3	Selection #4
				Gly	GGA	GGT
Val	GTA	GTT
			Ala	GCG	GCA
Ser	AGT	TCA
			Thr	ACG	ACT
Leu	TTG	CTT
			Pro	CCG	CCA

Codon-optimized versions of IV ("IV opt" (SEQ ID NO:22) and "IV optB" (SEQ ID NO:23)) and 9B8 ("9B 8 opt" (SEQ ID NO:24) and "9B 8 optB" (SEQ ID NO:25)) were generated and cloned into pF4Ag by methods known in the art. HEK293 cells were seeded at 15,000 cells/well in 96-well plates and grown overnight at 37 ℃. The following day, 100ng of plasmid DNA encoding a codon-optimized form in pF4Ag was used, using

LT1 Transfection Reagent (Mirus Bio) cells were transiently transfected into 6 well replicates and incubated overnight at 37 ℃. pGL4.13(Luc2/SV40) (Paguio et al, "pGL 4 Vectors: A New Generation of Luciferase Vectors" Promega Notes, 8) was also usedHEK293 cells were transfected with either 9:7-10(2005)) (or pGL4.73(hRL/SV40) (supra) to normalize for differences in transfection efficiency. 10 ng/transfection or 10% total DNA transfection was used. The medium was removed and the cells were lysed using 100. mu.L of lysis buffer containing 10mM CDTA, 150mM KCl, 10mM DTT, 100mM HEPES, pH 7.0, 35mM thiourea, and 0.5%

NP-9 (v/v). As follows

Luminescence of lysate samples was measured on an F500 photometer: for the hRL and OgLuc variants, luminescence of 10. mu.L of the lysis samples was determined using 50. mu.L of lysis buffer containing 20. mu.M substrate (native coelenterazine for hRL and PBI-3939 for the OgLuc variant). For Luc2(SEQ ID NOS: 28 and 29), firefly luciferase, using 50. mu.L of BRIGHT-GLO^TMLuciferase assay reagent (Promega Corp.) measures luminescence from 10. mu.L of lysed samples.

Fig. 32 shows the luminescence measured for lysates containing codon optimized versions of the OgLuc variants compared to hRL and Luc 2. The hRL and OgLuc variants were normalized to pgl4.13 and Luc2 to pgl4.73 using methods known in the art. As shown in fig. 32, Luc2 has approximately 14 times higher luminescence than hRL. The OgLuc variant has higher luminescence compared to Luc2 and hRL. The codon optimized versions of IV ("IV opt" and "IV optB") and 9B8 ("9B 8 opt") showed increased luminescence compared to the non-optimized versions.

As a result of the optimization, the "opt/opt A" form was expressed better in human HEK293 cells than its parental sequence, whereas the "opt B" form was less equally expressed in HEK293 cells than the parental sequence.

B.L27V

The L27V variant (SEQ ID NO:88) was optimized to minimize the occurrence of common vertebrate response elements (any Transcription Factor Binding Site (TFBS) in the Genomatx database). Three different optimized forms of the L27V variant were constructed:

L27V 01-form 1(SEQ ID NO:319) -the promoter component and all other unwanted sequence elements were removed by nucleotide substitution in addition to TFBS alone (see below for further details).

L27V 02-form 2-L27V01 was used as the starting, i.e. parent, sequence and TFBS was removed as soon as possible using high stringency matching criteria (higher stringency relates to better match with the binding site and will therefore find fewer matches than lower stringency). Two forms of L27V02, A (SEQ ID NO:322) & B ((SEQ ID NO:318)), were constructed. Both forms are constructed by selecting different codons for each form to remove unwanted sequence elements. Both formats were analyzed by searching with lower stringency for TFBS.

L27V 03-form 3(SEQ ID NO:325) -L27V02B (SEQ ID NO:318) was used as the starting sequence. The lower stringency TFBS matches are removed when possible. L27V03 was constructed as a very different codon to L27V 02A.

The following criteria were used to construct L27V optimized variants:

1. codon usage: preferably, the best two human codons are used for each amino acid (as was done for the IV variant) and the use of rare human codons (HS; encoding < 10% of the amino acids) is avoided (Table 19). If desired, rare E.coli Codons (EC) were used to remove unwanted sequence elements.

Watch 19

2. Removing unwanted sequence elements when possible

A. Restriction Enzyme (RE) site: the RE sites that may be useful for cloning are removed and should alternatively not be present in the Open Reading Frame (ORF).

B. Eukaryotic sequence elements: the splice donor and acceptor sites, splice silencer, Kozak sequence and polyadenylation sequence in the (+) mRNA strand were removed.

C. The vertebrate promoter component (PM) was removed (in the Genomatx catalog: vertebrate).

D. Vertebrate TFBS (in the Genomatx catalogue: vertebrate, universal core promoter elements and various other sequences) was removed when possible. This applies only to L27V optimizing forms 2 and 3, and not to form 1.

E. Coli sequence elements: the E.coli promoter was removed.

Mrna secondary structure: the strong secondary structure near the 5' end (high mRNA folding energy) (Zuker, Nucleic Acid Res.31(13):3406-3415(2003)) and other strong hairpin structures were removed.

Table 20 provides an alignment of sequences, with percent paired sequence identity ("()" indicating the number of nucleotide differences).

Watch 20

	L27V01	L27V02A	L27V02B	L27V03
					L27V00	99％(3)	97％	97％	94％
L27V01		98％(12)	98％	94％(32)
					L27V02A			99％(4)	95％(26)
L27V02B				96％

Example 26 Signal stability of OgLuc variant

A.15C1, 9B and IV

Signal stability of 15C1 was measured using PBI-3945 and 9B8 was measured using PBI-3889 and compared to IV. Coli containing plasmid DNA encoding 15C1, 9B8 or IV was cultured and induced in 8-well replication as previously described. Cells were lysed with lysis buffer containing 300mM HEPES pH 8.0, 0.3 Xpassive lysis buffer ("PLB"; Promega Corp. catalog No. E194A)0.3mg/mL lysozyme and 0.003U/. mu.L RQ1 DNase. Lysates were diluted 1:1000 in lysis buffer and used

The luminescence was measured with an F500 photometer. To 10. mu.L of the diluted lysate sample was added a solution containing 150mM KCl, 1mM CDTA, 10mM DTT, 100mM thiourea, 0.5%

NP-9(v/v) was measured immediately after 50. mu.L of "Glo" 0.5% TERGITOL assay buffer ("0.5% TERGITOL") and 20. mu.M of the novel coelenterazine PBI-3945 or PBI 3889.

The signal stability of the variants was determined by re-reading the plates every 30 seconds for a period of time after the addition of assay buffer to the samples. The signal half-life is determined from these measurements using methods known in the art. Mean signal half-lives were compared between variants and IV. 15C1 and 9B8 had a signal half-life of at least 30 minutes (fig. 33). Although 15C1 measured with PBI-3945 had higher luminescence at t-0, the signal decayed faster than the 9B8 variant measured with PBI-3889. At t 10 minutes, the light emission of 15C1 using PBI-3945 was the same as that of 9B8 using PBI-3889.

B.9B8 opt+K33N

The signal stability of the 9B8 opt + K33N variant was examined. Variant-containing E.coli lysates were prepared and analyzed as previously described, except that the assay buffer contained 0.25%

NP-9(v/v), 100mM MES pH 6.0, 1mM CDTA, 150mM KCl, 35mM thiourea, 2mM DTT, and 20. mu.M PBI-3939. Table 22 shows the signal half-life in minutes for the variants and indicates that the amino acid substitution L27V improves signal stability.

Table 22: signal stability of OgLuc variants in bacterial lysates

The signal activity and stability of the L27V variant (9B8+ K33N + L27V + T39T + K43R + Y68D; SEQ ID NOS: 88 and 89) was measured and compared to firefly luciferase and renilla luciferase. The L27V variant, Luc2 luciferase and Renilla luciferase were combined with

Fusion and in Escherichia coli expression. By using

Luciferase was purified as a purification tag according to the manufacturer's protocol (pFN 18A;

protein Purification System). 10pM of each purified luciferase (0.01% in the absence of phenol red)

DMEM) with equal volume of assay reagent (100 mM MES pH 6, 35mM thiourea, 0.5% for the L27V variant)

NP-9(v/v), 1mM CDTA, 2mM DTT, 150mM KCl, and 100. mu.M PBI-3939; the luciferase for firefly is ONE-GLO^TMThe Luciferase Assay System (Promega Corp.); and RENILLA-GLO for RENILLA luciferase^TMThe Luciferase Assay System (Promega Corp.) was mixed and luminescence monitored over time (3, 10, 20, 30, 45 and 60 minutes). FIGS. 34A-B show the high specific activity (FIG. 34A) and signal stability (FIG. 34B) of the L27V variant when compared to firefly luciferase and Renilla luciferase.

Example 27 enzyme kinetics of OgLuc variants

IV, 15C1, 9B8, 9F6 and 9A3

Enzymatic kinetic assays for measuring luminescence were performed using lysates of e.coli containing IV and IV variants 15C1, 9B8, 9F6, and 9A3 using methods known in the art. Cells were induced, lysed and diluted as described in example 26, except that the lysis buffer pH was 7.5. Two-fold serial dilutions of PBI-3939 in buffer were determined as previously described in example 26 using the diluted lysate assay. Fig. 35 shows the Km and Vmax values calculated using hyperbolic fitting of IV and variants 15C1, 9B8, 9F6 and 9 A3. Variants 9B8 and 9F6 had higher Km than IV, while the Km of the other variants was unchanged. Variants 15C1, 9B8, and 9F6 all had higher Vmax values compared to IV, while 8A3 had lower Vmax values.

15C1, which has the highest luminescence with PBI-3945, containing the amino acid substitution K33N, shows that K33N provides increased luminescence. The 9B8 variant was generated such that the additional substitution provided an increase in luminescence for the variant. Additional variants of 9B8 and 9F6 were generated to have amino acid substitutions for at least one of K33N or V38I ("9B 8+ K33N + V38I" and "9F 6+ K33N"). The variant 1D6 was used to enhance the importance of amino acid substitutions at positions 68, 72 and 75 for increased light output and stability. Fig. 36 shows Km and Vmax values calculated using hyperbolic fits for IV and variants 9B8, 9B8+ K33N + V38I, 9F6, 9F6+ K33N, and 1D 6. Although the actual Km values between fig. 35 and 36 for 9B8 and 9F6 are different, the general trend between variants is consistent.

The enzyme kinetics, i.e., Vmax and Km values were determined and compared as described previously for variants 9B8 opt and 9B8 opt + K33N, except using a mixture containing 1mM CTDA, 150mM KCl, 2mM DTT, 100mM MES pH 6.0, 35mM thiourea, 0.25%

NP-9(v/v), 10mg/mL 2-hydroxypropyl-. beta. -cyclodextrin and 20. mu.M PBI-3939 buffer were assayed for E.coli lysates. In that

Luminescence was measured on an F500 photometer. As shown in FIG. 37, the Vmax and Km values for 9B8 opt + K33N were higher than for 9B8 opt, indicating that the clone was brighter and had lower affinity for the substrate.

B.9B8 OPT + K33N variant

Determination of the enzyme kinetics values of the OgLuc variants as described previously, except that

The luminometer measures the luminescence. Three replicates were used for each variant. Table 23 shows the mean Km and Vmax (respectively "Km (+/-)" and "Vmax (+/-)" with standard deviation calculated using hyper.

Table 23: vmax value (RLU/0.5 sec) and Km (. mu.M) value of OgLuc variant

Sample (I)	Km	Km(+/-)	Vmax	Km(+/-)
					9B8	7.7	2.0	86,000,000	14,000,000
K33N	12.5	3.0	110,000,000	17,000,000
					T39T，Y68D	7.9	1.8	74,000,000	10,000,000
T39T，L27V，K43R	21.4	5.4	150,000,000	28,000,000
					L27V，T39T，K43R，Y68D	13.9	2.9	190,000,000	28,000,000
T39T，K43R，Y68D	10.5	2.8	140,000,000	25,000,000
					L27V，T39T，K43R，S66N	16.3	4.8	130,000,000	28,000,000
L27V，K43R，Y68D	13.7	4.3	130,000,000	28,000,000
					L27V，Y68D	10.2	3.0	97,000,000	19,000,000
L27VK43R，S66N	20.0	6.2	130,000,000	30,000,000

Example 28 protein stability of OgLuc variants

Since the stability of the luciferase protein is another factor affecting luminescence, the protein stability, i.e., thermostability, was determined.

A.15C1, 9B8, 9F6, 8A3 and IV

Lysates of E.coli containing 15C1, 9B8, 9F6, 8A3 or IV and E.coli expressing hRL (SEQ ID NOS: 30 and 31) were prepared from the induced cultures as previously described. Lysate samples were diluted 1:1000 with buffer containing 10mM HEPES pH 7.5 containing 0.1% gelatin. Diluted lysate (100 μ L) samples in two duplicate 96-well plates were incubated at 50 ℃. At various time points, the plates were placed at-70 ℃ (seventy degrees celsius). Each plate was thawed at room temperature, i.e. 22 ℃, for 10 minutes before measuring luminescence as previously described. Samples were assayed using native coelenterazine as a substrate (10. mu.L of each fused sample). Luminescence was measured for each time point plate immediately after addition of assay buffer. The half-life of the protein indicative of protein stability is calculated from the luminescence data for each time point using methods known in the art.

Table 24 shows the protein stability of variants 15C1, 9B8, 9F6 and 8A3 with time in minutes (hours) of 630.1(10.5), 346.6(5.8), 770.2(12.8) and 65.4(1.1), respectively. In contrast, hRL has a half-life of 9.6 minutes, while IV has a half-life of 27.2 minutes. Table 24 also shows that at 4 hours, 79%, 61%, and 80% of 15C1, 9B8, and 9F6, respectively, retained activity.

Table 24: protein stability of OgLuc variants at 50 ℃

Sample (I)	Half-life (minutes)	Half-life (hours)	t is the remainder at 4 hours%
				Genus Renilla	9.6
IV	27.2
				15C1	630.1	10.5	79％
9B8	346.6	5.8	61％
				9F6	770.2	12.8	80％
8A3	65.4	1.1

1D6, 9B8, 9B8+ K33N + V38I, 9F6, 9F6+ K33N, and IV

Lysates of E.coli containing 1D6, 9B8, 9B8+ K33N + V38I, 9F6, 9F6+ K33N or IV were prepared from the induced cultures and luminescence was determined as previously described. In this example the protein stability, i.e. the thermostability of the lysate, was determined as described above. Fig. 38 shows the half-life of the variant in minutes at 50 ℃ and the luminescence of the sample measured at the beginning of the incubation period, i.e. t-0, using native coelenterazine as substrate. The difference between variants 9B8+33+38 and 9F6 was an amino acid substitution, L27V, indicating that the amino acid substitution increased stability. Increased "activity/expression" substitutions at positions 68, 72 and 75 improve stability. Fig. 38 shows that the provided K33N provides the variant 9F6 with higher thermal stability and the variant 9B8 has higher light output and stability than the variant 1D 6. The differences between the two variants, i.e., 9B8 containing additional amino acid substitutions F68Y, L72Q, and M75K, indicate the importance of the three substitutions.

In addition to thermal stability, structural integrity, stability and solubility as determined by expression can also affect luminescence. As a way to further test the structural integrity of the improved variants, those based on pF4Ag (i.e., no HT7) will be includedOgLuc variant N166R (previously described in U.S. application Ser. No. 12/773,002 (U.S. published application No. 2010/0281552)), C1+ A4E, IV, 9B8, and 9F6 of KRX E.coli was cultured in Luria Broth (LB) to OD 6 at 37 deg.C₆₀₀0.6 and overexpression was then induced by addition of rhamnose (0.2% final concentration). Then, the repeatedly induced culture was incubated at 25 ℃ or 37 ℃ for 17 hours, at which time the total (T) fraction and the lysis (S) fraction were prepared and purified by SDS-PAGE using simple blue^TMSafesain (Invitrogen) was analyzed by staining the gel (FIGS. 39A-B). hRL and Luc2 were used as controls.

The OgLuc variant, hRL and Luc2 were well expressed and soluble when induction occurred at 25 ℃ (FIG. 39A; note that for the approximately 19kDa black band in the "soluble" fraction of the OgLuc variant, the N166R variant was removed, and for the approximately 36 kDa band and the 64kDa band in the "soluble" fraction of hRL and Luc2, respectively). In contrast, while C1+ A4E, IV, 9B8 and 9F6 performed well at 37 ℃ (significantly better than either hRL or Luc2 as shown in the "total" fraction), only the 9B8 and 9F6 variants were soluble when increased induction temperatures were used (see fig. 39B; note about 19kDa black bands in the "soluble" fraction for 9B8 and 9F 6). These results are consistent with the thermal stability data shown in table 24 and fig. 38.

C.9B8 OPT and 9B8 OPT + K33N

The thermostability of the variant variants 9B8 opt and 9B8 opt + K33N were compared. Coli lysates containing variants 9B8 opt or 9B8 opt + K33N were prepared and analyzed as previously described except for the following differences: lysates were diluted 1:100 in the lysis buffer previously described and repeatedly diluted lysates were incubated at 60 ℃ in a temperature cycler. Aliquots were removed at different time points and placed on dry ice to freeze the samples. The frozen lysate was thawed at 22 ℃ and used as a medium containing 20mM CDTA, 150mM KCl, 10mM DTT, 20. mu.M PBI-3939, 100mM HEPES pH 7.0, 35mM thiourea, and 0.1%

NP-9(v/v) was measured in a buffer. In that

Luminescence was measured on a luminometer (Promega Corp.). Fig. 40A shows the light output time course of the natural log values of luminescence measured in RLU units over time (in minutes). As shown in figure 40B, 9B8 opt + K33N had a half-life of 6.8 hours at 60 ℃ that was longer than the 5.7 hour half-life of 9B8 opt.

Table 25 shows the thermal stability ("T") of 9B8 opt and 9B8 opt + K33N at 60 ℃. (see_1/2(60 ℃) "), and luminescence (" RLU ") data at the beginning of the incubation time (i.e., t ═ 0). 9B8 opt + K33N was more stable and had approximately 1.8 times higher brightness than 9B8 opt, indicating that the amino acid substitution K33N provided higher light output and higher thermal stability.

Table 25: thermal stability and luminescence data for 9B8 opt and 9B8 opt + K33N

D.9B8+ K33N variants

The thermostability of the variants at 60 ℃ was checked as before except that the assay buffer contained 100mM MES pH 6.0 instead of HEPES. Table 26 and figure 41 show the half-life in hours of the variants at 60 ℃. The data indicate that the amino acid substitution L27V improved thermostability.

Table 26: thermostability of OgLuc variant at 60 ℃

Variants 9B8 and V2(9B8+ K33N + T39T + K43R + Y68D) were also screened in HEK293 cells to determine their stability. The variants were cloned into pF4Ag and transfected into HEK293 cells (15,000 cells/well) as previously described. After transfection, the cells were lysed in assay reagent (as described previously; no PBI-3939) and luminescence was measured using assay reagent containing 20. mu.M PBI-3939. 9B8 has a half-life of 5.2 hours, while V2 has a half-life of 16.8 hours. This is consistent with the half-lives found for these variants in E.coli (Table 26).

L27v variants

The activity of the L27V variant (9B8+ K33N + L27V + T39T + K43R + Y68D) was evaluated at different pH and different salt conditions. It has been previously shown that 9B8 and 9B8+ K33N have similar stability at pH 6 and pH 7 (data not shown). To evaluate activity under different salt conditions, 50 μ L of assay buffer containing 20 μ M PBI-3939 and varying amounts of KCl or NaCl were mixed with 50 μ L HEK293 cells transiently transfected with L27V (pF4 Ag). Luminescence was measured and percent activity (ratio of luminescence to no salt) was determined (fig. 42B). To evaluate activity at different pH, a composition containing 100mM citrate, 100mM MES, 100mM PIPES, 100mM HEPES, 100mM TAPS, 0.5%

NP-9(v/v)，0.05％

DF 204, 1mM CDTA, and 1mM DTT reagent, titrated for different pH values. 362pM L27V in assay reagent was mixed with substrate 100. mu.M PBI-3939 and luminescence was measured (FIG. 42A).

Example 29 gel filtration chromatography of OgLuc variants

C1+ A4E and 9B8

The gel filtration analysis was used to verify the desired molecular weight of the purified OgLuc protein based on theoretical values and thereby determine its oligomeric state. A comparison between the relative hydrodynamic volumes of the OgLuc variants C1+ A4E and 9B8 was performed by gel filtration chromatography. To perform this analysis, the nucleotide sequences of the OgLuc variants C1+ A4E and 9B8 were cloned into HQ-Tagged

HQHQ N-terminally tagged protein overexpressed in E.coli KRX cells was constructed in Vector (Promega Corp.). Using HISLINK^TMProtein Purification System (Promega Corp.) the overexpressed proteins were purified according to the manufacturer's instructions. Each individual standard was analyzed by gel filtration chromatographyA sample of the sample protein was carried out on an Agilent 1200HPLC at 24 ℃ using a Superdex 2005/150 GL column (GE Healthcare) with a flow rate of 0.25 mL/min. The mobile phase (i.e., running buffer) consisted of 50mM Tris and 150mM NaCl, pH 7.5. Protein elution was monitored at 214 and 280 nm. A standard calibration curve was generated using: 1) ovalbumin, 43kDa (GE healthcare), 2) carbonic anhydrase, 29kDa (Sigma) and 3) myoglobin, 17 kDa (Horse Heart, Sigma). The molecular weight of the purified protein was calculated directly from the calibration curve.

The relative elution of protein observed with this column at 7.98 min was ovalbumin, carbonic anhydrase at 8.65 min, 9B8 at 8.97 min, and myoglobin at 9.06 min (fig. 43A-B). As shown in fig. 43B, 9B8 eluted as a 21kDa protein (predicted molecular weight of about 19kDa), indicating that 9B8 was present as a monomer, while C1+ A4E eluted at about 4.3 minutes (fig. 43A), indicating that C1+ A4E was expressed and present as a multimer, e.g., possibly as a tetrameric complex or larger.

L27V variants

To demonstrate that the OgLuc variant L27V exists in the monomeric state, the desired molecular weight of the purified L27V protein was verified based on theoretical values using gel filtration analysis and thus its oligomeric state was determined. Relative hydrodynamic volume of L27V was performed by gel filtration chromatography. To perform this analysis, the nucleotide sequence of the L27V variant was cloned into

Vector pFN18A (Promega Corp.) to construct a Vector that is overexpressed in E.coli KRX cells

-terminally labeled proteins. By using

Protein Purification System (Promega Corp.) the overexpressed proteins were purified according to the manufacturer's instructions. Samples of each individual standard and sample protein were analyzed by gel filtration chromatography at 24 ℃ at A gilent 1200 HPLC was performed using a Superdex 2005/150 GL column (GE Healthcare) with a flow rate of 0.25 mL/min (FIG. 56). The mobile phase (i.e., running buffer) consisted of 50mM Tris and 150mM NaCl, pH 7.5. Protein elution was monitored at 214 and 280 nm. A standard calibration curve was generated using: 1) ovalbumin, 43kDa (GE Healthcare), 2) myoglobin, 17kDa (Horse Heart, Sigma) and 3) ribonuclease, 14kDa (Bovine pancreas). As shown in fig. 44, the L27V variant eluted as a 24kDa protein (predicted molecular weight of about 19kDa), indicating its presence as a monomer.

Example 30 protein expression levels of OgLuc variants

Iv, 8a3, 8F2, 9B8, 9F6 and 15C1

Normalization of protein expression provides information about possible differences in specific activity. To provide a means to quantify protein expression, the OgLuc variant was cloned into the pF4Ag vector containing C-terminal HT7 as previously described to generate the OgLuc variant-HT 7 fusion protein. The following fusion proteins were generated: IV-HT7(SEQ ID NOS: 48 and 49), 8A3-HT7(SEQ ID NOS: 34 and 35), 8F2-HT7(SEQ ID NOS: 50 and 51), 9B8-HT7(SEQ ID NOS: 36 and 37), 9F6-HT7(SEQ ID NOS: 38 and 39), and 15C1-HT7(SEQ ID NOS: 52 and 53). Coli containing the OgLuc variant-HT 7 fusion was grown and induced as previously described. With 100. mu.L of 10 XFASTBREAK ^TMCell lysis reagent (Promega Corp.) lysed 900 μ L of Cell culture. Will be provided with

TMR-ligand (Promega Corp.) was added to each bacterial lysate sample to obtain a final concentration of 0.5 μ M. At room temperature according to the manufacturer's instructions

The bacterial lysates were incubated for 30 min with TMR-ligand. Using 1X FASTBREAK^TMmu.L of each sample was diluted, i.e., 10. mu.L of sample was added to 10. mu.L of 1X FASTBREAK^TM. For each sample 15. mu.L of lysate and 15. mu.L of 1:1 dilution were analyzed by SDS PAGE. Resolving labeled fusion eggs by SDS-PAGEWhite, using simple blue^TMSafesain (FIG. 45A) and fluorescent imager (GE Healthcare Typhoon). The bands were quantified using Imagequant software (GE Healthcare). FIG. 45B shows the band volumes measured from FIG. 45A for IV-HT7 ("IV"), 15C1-HT7 ("15C 1"), 9B8-HT7 ("9B 8"), 9F6-HT7 ("9F 6"), and 8F2-HT7 ("8F 2"), normalized to IV-HT 7. The data show that the IV variants are well expressed compared to IV.

B.9B8 opt, V2 and L27V

The expression levels and solubilities of 9B8 opt, V2 and L27V were compared. The three variants were used to transform E.coli KRX cells in the pF4Ag background. The resulting clones were used for expression experiments, in which individual clones grown overnight at 30 ℃ diluted 1:100 in LB were grown to OD ₆₀₀About 0.5 and then induced with 0.2% rhamnose at 25 ℃ for 18 hours. Then at 0.5X FASTBREAK^TMCells were incubated in the presence of lysis reagent (Promega Corp.) at room temperature for 30 minutes and the resulting lysates were stored at-20 ℃. After slow thawing on ice, the soluble fraction was prepared by high speed centrifugation at 4 ℃ for 10 minutes. The expression of the crude total (T) and soluble (S) fractions was then analyzed by SDS-PAGE + simple blue staining (fig. 46A) and by measuring luminescence. For luminescence measurements, 50. mu.L of soluble lysate in a 96-well microtiter plate was mixed with 50. mu.L of assay reagent (previously described; 40. mu.L of PBI-3939) and used

F500 multiple assay plate reader measures luminescence. These results indicate that the three variants are ranked as L27V with respect to their expression level and solubility>V2>9B8opt。

Example 31 Brightness of OgLuc variants expressed in mammalian cells

A.IV and 9B8

The brightness of IV and 9B8 variants in pF4Ag vector (i.e., no HT7) was evaluated in HEK293 cells. hRL was used as a control. Briefly, HEK293 cells seeded at 15,000 cells/well in 96-well plates were used with plasmids containing sequences encoding different variants and/or control sequencesOf DNA

LT1 for transient transfection. Cells were cultured, lysed and processed as described in example 25. Cells were co-transfected with pGL4.13(Promega Corp.) as a transfection control (10 ng/total DNA transfected or transfected 10% was used). Luminescence was measured as described previously using native coelenterazine as a substrate for hRL or PBI-3939 as a substrate for the OgLuc variant. The OgLuc variant data was corrected for transfection efficiency using Luc2 luminescence (i.e., luminescence measured after addition of fluorescein substrate). The OgLuc variants IV and 9B8 had higher luminescence compared to hRL (fig. 47).

To compare brightness on a per-molar basis in mammalian cells, the HT7 fusion protein of variant 9B8 described in example 30 ("pF 4Ag-OgLuc-9B8-HT 7") was analyzed and compared to the C-terminal HT7-hRL fusion protein ("pF 4Ag-renilla-HT 7") and the C-terminal HT7-Luc2 fusion protein ("pF 4Ag-Luc2-HT 7"). HEK293 cells (15,000) were seeded and grown overnight at 37 ℃. These cells were transfected with 100ng of DNA from pF4Ag-Renilla-HT7, pF4Ag-Luc2-HT7 or pF4Ag-OgLuc-9B8-HT7 and grown overnight at 37 ℃. The medium was removed and the cells lysed as previously described. Luminescence (RLU) was determined for 10. mu.L of each sample using 50. mu.L BRIGHT-GLO for Luc2 ^TMLuc2, 50. mu.L of 20. mu.M native coelenterazine for hRL, and 50. mu.L of 20. mu.M PBI-3939 for variant 9B 8.

Lysates from 6 wells were mixed and used as described in example 30

The TMR-ligand is labeled. The labeled fusion protein was resolved by SDS-PAGE and subjected to fluorescence imaging (GE Healthcare Typhoon). Band densities were determined to quantify the relative moles present for each luciferase and RLU values were normalized for each sample by calculating band concentrations to normalize the expression levels for each protein, i.e., RLU was normalized using TMR marker quantitation (fig. 48). The 9B8 variant was approximately 15 times brighter than Luc2 on a molar-to-molar basisAnd is about 100 times higher than hRL. The data represent the difference in specific activity.

B.9B8 opt and 9B8 opt + K33N

The brightness of variants 9B8 opt and 9B8 opt + K33N expressed in HEK293 cells was measured and compared as described for the HT 7-free variants in example 31. HEK293 cells were transfected with 30 and 100ng plasmid DNA containing variant DNA. Cells were grown and induced as described in example 31, except using a medium containing 1mM CTDA, 150mM KCl, 2mM DTT, 100mM MES pH 6.0, 35mM thiourea, 0.25%

NP-9(v/v), and 10mg/mL of 2-hydroxypropyl-. beta. -cyclodextrin in lysis buffer. Lysates were assayed in lysis buffer containing 20 μ M PBI-3939 and assayed in

GENIOS^TMLuminescence was measured on a Pro luminometer. As shown in figure 49, 9B8 opt + K33N had higher luminescence in HEK293 cells compared to 9B8, which is consistent with the bacterial expression data in table 25 and figure 29.

C.9B8+ K33N variants

The brightness of the variants expressed in HEK293 cells and NIH3T3 cells was measured as previously described. The luminescence of the variants was normalized with respect to the luminescence generated by 9B8 opt (table 27).

Table 27: increase in luminescence by OgLuc combinatorial variants in NIH3T3 cells and HEK293 cells

Sample (I)	HEK293	NIH3T3
			9B8	1.0	1.0
K33N	1.8	1.5
			T39T,Y68D	1.9	1.5
T39T,L27V,K43R	1.3	0.9
			L27V,T39T,K43R,Y68D	1.6	1.6
T39T,K43R,Y68D	1.9	1.9
			L27V,T39T,K43R,S66N	1.3	1.2
L27V,K43R,Y68D	1.6	1.5
			L27V,Y68D	1.7	1.4
L27V,K43R,S66N	1.2	1.0

D.L27V

Comparison of the luminescence of the L27V variant was performed with firefly luciferase alone and firefly luciferase as a fusion. HEK293 cells and HeLa cells were seeded into wells of 12-well plates at 15,000 and 10,000 cells/well, respectively, and 5% CO at 37 ℃%₂Incubation was performed overnight. Cells were then transfected with serial dilutions of pF4Ag containing L27V or Luc 2. 20ng of pGL4.13(Promega Corp.) were co-transfected with L27V, and 20ng of pGL4.73(Promega Corp.) were co-transfected with Luc2 to be used as vector DNA for L27V or Luc2 plasmid DNA. Then use

LTI transfection reagent plasmid DNA was transfected into cells (6 replicates per dilution for each cell type) according to the manufacturer's instructions. Then 5% CO at 37 deg.C₂Cells were incubated for 24 hours.

After transfection, the medium was removed from the cells and added at 0.5%

NP-9(v/v) in 100. mu.L PBS and shaken at room temperature for 10 minutes. Using ONE-GLO^TMThe Luciferase Assay System (Promega Corp.; Luc2) or the Assay reagents (example 22H containing 20. mu.M PBI-3939; OgLuc). Luminescence was measured as previously described for HEK293 cells (fig. 50A) and HeLa cells (fig. 50B).

A comparison of L27V and Luc2 as fusion partners was performed as described above. Mixing L27V and Luc2 with pF4Ag

And (3) protein fusion. FIGS. 50C-D show luminescence measured with different fusions in HEK293 cells (FIG. 50C) and HeLa cells (FIG. 50D).

In addition to measuring luminescence, protein tables were also analyzedSo as to achieve the purpose. Transfection was performed as described above. After transfection, the medium was removed from the cells and the cells were washed in 1X PBS. Adding a solution containing 1 μ M

TMR ligand (Promega Corp.) and 20U of DNase I in 100. mu.L of 0.1 Xmammalian lysis buffer (Promega Corp.) and incubating the cells at room temperature for 45 min with slow shaking. The cell samples were then frozen at-20 ℃. For protein analysis, 32.5 μ L of 4X SDS loading dye was added to each sample and the samples were heated at 95 ℃ for 2 minutes. A10 μ L sample was then loaded onto an SDS-PAGE gel and imaged on a Typhoon Scanner as previously described (FIG. 50E).

Example 32-Brightness of purified OgLuc variants compared to firefly luciferase

The 9B8 OgLuc variant was overexpressed and purified as described in example 33. The reaction was performed between the diluted enzyme and substrate using the following 2X buffer/assay reagents: 100 mM MES pH 6.0, 1mM CDTA, 150mM KCl, 35mM thiourea, 2mM DTT, 0.25%

NP-9(v/v)，0.025％

DF 204, 10 mg/mL 2-hydroxy- β -cyclodextrin, and 20 μ M PBI-3939. The final assay concentrations of purified enzyme and substrate were 0.5pM and 10. mu.M, respectively. In parallel, the diluted purified firefly luciferase (i.e.,

reaction between Recombinant Luciferase (Promega Corp.) and fluorescein. The assay buffer/reagent for the firefly luciferase reaction is BRIGHT-GLO^TMAnd final assay concentrations of 0.5pM enzyme and 500. mu.M fluorescein. Since the buffer/reagent is known to provide "glow" kinetics for each reaction, a 15 minute time point was used to collect luminescence data. The results from this experiment showed 9B8 using PBI-3939(19,200RLU)opt Brightness ratio Using BRIGHT-GLO^TM(2,300 RLU)

The Recombinant Luciferase was about 8-fold higher.

Example 33 inhibition assay

To determine the sensitivity of the OgLuc variants to off-target interactions, 9B8 and L27V variants were screened for activity against a library of LOPAC (a library of pharmacologically active compounds). The LOPAC 1280 library (Sigma) was prepared by diluting the compounds to 1mM in DMSO. On the day of assay, compounds were diluted to 20 μ M in 1X PBS and 10 μ L was transferred to 96-well white plates. To each well was added 10% in a Glo Lysis Buffer (Promega Corp.) ^-410 μ L of purified 9B8, L27V or firefly luciferase (Lu2) diluted and incubated at room temperature for 2 minutes. To the sample was added 20. mu.L of assay reagent (1mM CDTA, 150 mM KCl, 2mM DTT, 100mM MES pH 6.0, 35mM thiourea, 0.5%

NP-9(v/v) and 60. mu.M PBI-3939), incubated for 3 minutes and incubated at

GENIOS^TMLuminescence was measured on a Pro luminometer. For the determination of firefly luciferase, BRIGHT-GLO was used according to the manufacturer's protocol^TMAssay reagents (Promega Corp.). As a negative control, 8 wells of each plate contained 1X PBS + 2% glycerol. As positive controls, 8 wells of each plate contained 2mM suramin in 2% DMSO or 2mM luciferase inhibitor 1(Calbiochem) in 2% DMSO. Suramin was identified as an inhibitor of OgLuc in the primary screen of the LOPAC library (i.e., screening the LOPAC library using the 9B8 variant with a lower substrate concentration of 20 μ M).

The results in figure 51 indicate a generally low frequency of off-target interactions between the LOPAC library and the compounds in L27V. This suggests the potential use of L27V as a screening tool for large libraries (e.g., high throughput screening) that include different chemicals and therapeutic candidates based on the form of living cells.

To further examine the inhibition, purified 9B8 and L27V were screened against different concentrations of suramin (Sigma S-2671) and Tyrphostin AG 835 ("Tyr AG 835") (Sigma T-5568) (FIGS. 52A-C). FIGS. 52E-D show the chemical structures of Suramin and Tyr ag 835, respectively. Purified 9B8 and L27V were prepared as described above. Serial dilutions of the inhibitor (0, 2. mu.M, 6. mu.M, 20. mu.M, 60. mu.M, 200. mu.M and 2mM) were prepared in 1 XPBS containing 2% DMSO. To the wells of a 96-well white assay plate, 10 μ L of diluted enzyme and 10 μ L of diluted inhibitor were added and incubated for 2 minutes at room temperature. Add 20. mu.L of assay reagent (as described above) and add

Luminescence was measured on a 96 photometer (FIGS. 52A-C). Fig. 52A-B show dose response curves for 9B8 and L27V for suramin (fig. 52A) and tyrag 835 (fig. 52B). FIG. 52C shows the half maximal Inhibitory Concentrations (IC) for 9B8 and L27V suramin and Tyrag 835₅₀). The data indicate that L27V is a robust reporter useful as a screening tool for large libraries of different chemical and/or therapeutic candidates.

Example 34 resistance to non-specific protein interactions

1. Purified 9B8 and L27V enzymes were added in buffer (1 XPBS, 1mM DTT and 0.005%)

CA-630) was diluted 1:10 to 200. mu.L into a PCR tube. The samples were incubated at 60 ℃ with one set of dilutions of each variant being transferred to-70 ℃ at 0, 2, 4 and 6 hours.

To analyze activity, the samples were thawed to room temperature in a water bath. Add 50. mu.L of assay reagent (100. mu.M PBI-3939 as described previously) and add

Luminescence was measured for 30 minutes per minute on an F500 plate reader. Using 1x10⁶And 1x10⁷Diluted mean luminescenceActivity was calculated (fig. 53).

2. To demonstrate the reactivity of the OgLuc variant towards plastics, purified 9B8 and L27V were contacted with polystyrene plates and their activity was measured.

At 60, 40, 20 and 0 minutes, the phenol red-free content was 0.1%

50 μ L of purified 9B8(45.3pM) and L27V (85.9pM) in DMEM was placed in the wells of a 96-well polystyrene microtiter plate. To the sample was added 50 μ L of assay reagent containing 20 μ M PBI-3939 (as described above) and incubated for 5 minutes at room temperature. Luminescence was measured as previously described and percent activity determined (fig. 54; ratio of luminescence to time 0).

Example 35 post-translational modification

To determine whether the OgLuc variants undergo any post-translational modifications when expressed in mammalian cells, the 9B8 and L27V variants were expressed in mammalian cells and e.

The 9B8 and L27V variants were used as N-termini in HEK293 cells and E.coli KRX (Promega Corp.) cells

Fusions (pFN 18K for E.coli cells; pFN21K for HEK293 cells) were expressed and utilized

The Protein Purification System (Promega Corp.) was purified according to the manufacturer's instructions. Approximately 5 picomoles of purified enzyme were analyzed via LC/MS using a C4 column (Waters Xbridge BEH300, 3.5 μm) connected to an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific). Data were obtained from 600-. Both purified enzymes had an experimentally determined mass of 19,666Da compared to the calculated mass of 19,665Da for the unmodified OgLuc variant (i.e., in the absence of any post-translational modifications).

Example 36 evaluation of OgLuc variants as post-transcriptional reporters

A.IV

The use of the OgLuc variants as a post-transcriptional reporter was examined. To generate a post-transcriptional reporter of cAMP, hRL and IV were subcloned into a modified pGL4 vector (Promega Corp.) containing the barnase sequence replaced by the DNA fragment of interest using methods known in the art. The leader sequence of pGL4 was modified to contain a minimal promoter and a cAMP response element (CRE; SEQ ID NO:96) so that upon stimulation with a cAMP agonist such as Forskolin (FSK), cells that accumulate cAMP activate the reporter and produce luminescence. In this experiment, HEK293 cells were transfected as described in example 25 using 2ng DNA of either the hRL or IV transcription reporter constructs. At 24 hours post-transfection, cells were treated with 100 μ M FSK. Cells not treated with FSK were used as controls. After 6 hours, reporter reagents were added to the treated cells and control cells. For hRL, the reporter reagent is Renilla-Glo ^TMReagent (Promega Corp.). For IV, the reporter reagent contained 1mM CDTA pH 5.5, 150mM KCl, 10mM DTT, 0.5%

NP-9(v/v), 20. mu.M coelenterazine-h and 150mM thiourea. After 10 minutes, in

Read luminescence on flash (thermo scientific).

Figure 55 shows the normalized luminescence of HEK293 cells containing FSK treated ("+ FSK") or FSK untreated ("-FSK") hRL ("renilla") or IV transcription reporters. Responses were determined by dividing luminescence from treated cells (+ FSK) by luminescence from control cells (-FSK), i.e., fold induction or fold increase ("fold") of luminescence. As shown in figure 55, for hRL, the response was <50, whereas for IV it was >300, indicating the use of IV as a transcriptional reporter.

B.9B8 and 9B8 opt

Also checkThe use of variants 9B8 and 9B8 opt as transcription reporters was demonstrated and compared to the hRL and Luc2 transcription reporters with the following modifications as previously described for the IV transcription reporter. Transcriptional reporters containing cAMP for variants 9B8 or 9B8 opt were generated as described above. After 6 hours of FSK induction, the medium was removed from the cells and replaced with 100 μ L of lysis buffer as described in example 25 to generate a lysate. Luminescence of lysates of transfected cells treated with or without FSK was determined as described in example 25. Using 50. mu.L of BRIGHT-GLO ^TM Luciferase assay reagent 10. mu.L of Luc2 lysate was assayed. mu.L of hRL lysate was assayed with 50. mu.L of lysis buffer containing 20. mu.M native coelenterazine. mu.L of variant 9B8 and 9B8 opt lysates were assayed using 50. mu.L of lysis buffer containing 20. mu.M PBI-3939.

Figure 56 shows normalized luminescence of HEK293 cells containing 9B8, 9B8 opt, hRL or Luc2 transcription reporters treated with FSK ("induced") or not treated with FSK ("basal"). The response was determined by dividing the induced luminescence by the substrate luminescence, i.e., the fold induction or fold increase ("fold") of the luminescence (fig. 56). Although fold induction values were similar for each reporter except Luc2, the luminescence generated by the induced 9B8 opt transcriptional reporter was about 2.5 log higher than the induced renilla transcriptional reporter and about 1.5 log higher than the Luc2 transcriptional reporter. FIG. 56 shows the use of 9B8 and 9B8 opt as transcription reporters.

C.9B8 opt and 9B8 opt + K33N

Variants 9B8 opt and 9B8 opt + K33N were compared in a split transcription reporter assay. Variant 9B8 opt + K33N was cloned into a cyclic AMP response element (CRE) -containing pgl4.29 vector (Promega Corp.) using methods known in the art. The 9B8 opt + K33N transcriptional reporter was tested and compared to the 9B8 opt transcriptional reporter in HEK293 cells as described above. HEK293 cells were transfected with 30 and 100ng plasmid DNA containing variant transcription reporter forms. Cells were induced with FSK for 5 hours before measuring luminescence. Using a mixture containing 1mM CTDA, 150mM KCl, 2mM DTT, 100mM MES pH 6.0, 35mM thiourea, 0.25%

NP-9(v/v) and 10mg/mL 2-hydroxypropyl-. beta. -cyclodextrin in lysis buffer lysed cells. In that

GENIOS^TMLuminescence was measured on a Pro luminometer. Lysates were assayed with lysis buffer containing 20 μ M PBI-3939. Figure 57 shows normalized luminescence (transfection corrected) of HEK293 cells expressing 9B8 opt or 9B8 opt + K33N transcriptional reporter constructs treated with FSK ("induced") or not treated with FSK ("basal"). As shown in figure 57, the fold induction of 9B8 opt was 360 when transfected with 30ng of DNA and 109 when transfected with 100ng, while the fold induction of 9B8 opt + K33N was 275 and 147, respectively. K33N provided a higher response when transfected with higher amounts of DNA.

D.L27V

1. L27V was cloned into a reporter vector containing a CRE, NFkB or HSE (heat shock element) responsive element as described in C of this example. The reporter constructs were then transfected into HEK293 cells or HeLa cells as previously described. Cells were induced using FSK for CRE, TNF α for NFkB or 17-AAG for HSE. Luminescence was measured as previously described using assay reagents containing 20 μ M PBI-3939 (FIGS. 58A-C). All reporter constructs were validated in HEK293, HeLa, NIH3T3, U2OS and Jurkat cell lines (data not shown).

2. L27V02 and L27V02P (containing PEST sequences; SEQ ID NO:323) were cloned into a reporter vector (pGL4.32 based) as described in C of this example. Other OgLuc variants of PEST-containing sequences include L27V01-PEST00 and L27V03-PEST02 (SEQ ID NOS: 320 and 326, respectively). The reporter construct was then transfected into HEK293 cells as previously described. Cells were then induced using FSK and luminescence was measured as previously described using assay reagents containing 20 μ M PBI-3939 (fig. 59A-B). Different other reporter constructs were also constructed and tested in different cell lines (fig. 59C). Figure 59A shows the full dose response to CRE cells in HEK293 cells. Fig. 59B summarizes fig. 59B. FIG. 59C summarizes the data in FIGS. 59A-B and shows the same data types for NFkB responder elements. Both CRE and NFkB reporter constructs were examined in HEK293, HeLa, HepG2, Jurkat, ME180, HCT116 and U2OS cell lines.

3. By using

HD (Promega Corp.) the secretory construct pNFkB-L27V (SEQ ID NO: 463) was used according to the manufacturer's instructions&464; wherein the IL-6 secretion sequences (SEQ ID NOS: 461 and 462) replace the native OgLuc secretion sequence SEQ ID NO: 54), Metridia longa (Clontech), pNFkB-L27V (native secretion sequence; 465 and 466) or firefly luciferase (Luc 2; pGL4.32) plasmid DNA transfection of HEK293 cells (0.9X 10 in T25 flasks) ⁶Individual cells). At 37 deg.C, 5% CO₂Cells were incubated for 8 hours and then trypsinized in 0.5mL TrypLE (Invitrogen). The lysate was then resuspended in 8mL DMEM containing 10% FBS, 1X NEAA and 1X sodium pyruvate. Then 100. mu.L of the reselected sample was added to the wells of a 96-well plate and 5% CO at 37 ℃%₂Incubate for 16 hours.

After incubation, the medium was removed from the cells and replaced with 100 μ L of fresh medium with or without TNF α (serial dilution). To measure secretion, 5 μ L of medium (triplicate) was removed from the cells at 3 and 6 hours, increased to 50 μ L using PBS and mixed with 50 μ L of assay reagent (100 μ M PBI-3939 as described previously). Luminescence was measured at 0 and 10 minutes as previously described (fig. 60).

To measure luciferase Activity of Metridia elongata, Ready-To-Glow was used according To the manufacturer's protocol^TMThe Secreted Luciferase System (Clontech). Briefly, 5. mu.L of Ready-to-Glow was added^TMReagents were added to 5. mu.L of sample and 45. mu.L of PBS. Luminescence was measured immediately after reagent addition (fig. 60).

L27v optimized variants.

According to manufacturer's scheme

Preparation of HDPlasmid DNA for transfection (pGL4.32-L27V00, pGL4.32-L27V01, pGL4.32-L27V02, pGL4.32-L27V03 and pGL4.13). pGL4.32 vector (Promega Corp.) contains NF-. kappa.B response elements. The Luc2P sequence in the vector was replaced by a codon optimized sequence of L27V. pGL4.13 vector (Promega Corp.) contains Luc2 gene driven by SV40 promoter.

Then 300. mu.L of the DNA transfection mixture was mixed with 6mL of HeLa cell suspension (2X 10)⁵Individual cells/mL) were mixed, homogenized, and 100 μ L was seeded into wells of a 96-well plate. Then 5% CO at 37 ℃₂Cells were incubated overnight. After incubation, 10 μ L of 10 XrhTNF α in BSA containing DPBS was added to the wells and 5% CO at 37 ℃%₂Incubate for 4.5 hours. 6 wells were used as vehicle only. The cells were then allowed to equilibrate for 20 minutes at room temperature, and then 100 μ L of assay reagent (containing 100 μ M PBI-3939 as previously described) was added. Addition of 100. mu.L of ONE-GLO to Luc2 expressing cells or vehicle treated cells alone^TMA luciferase assay reagent. Luminescence was measured 12 minutes after addition of assay reagents as previously described. FIGS. 61A-B show absolute luminescence, FIGS. 61C-D show normalized luminescence and FIGS. 61E-F show fold response.

Example 37 OgLuc variants in transcription reporter assays

To demonstrate the ability of the OgLuc variants of the invention to serve as transcription reporters, the OgLuc variant 9B8 opt was used as a transcription reporter in forward, reverse, and bulk transfections. These transfections were chosen according to the manufacturer's protocol as they represent the method commonly used in transient expression of gene transcription reporters.

Forward transfection

Transcription reporters containing CAMP Response Element (CRE) and 9B8 opt or 9B8 opt (9B8 opt-P) further including PEST protein degradation sequences were prepared in pGL4.29(Promega Corp.) backbone, i.e., the luc2P gene of pGL4.29 vector was replaced with 9B8 opt (SEQ ID NO:24) or 9B8 opt-P (SEQ ID NO: 65). pgl4.29 was used as a control/reference point.

HEK293 cells were seeded at 15,000 cells/well in 6 96-well tissue culture plates. DMEM + 10% FBS +1X at 100. mu.LCells were cultured in non-essential amino acids (NEAA) and incubated overnight at 37 ℃. Cells were transiently transfected with 10ng or 100ng plasmid DNA per well of pGL4.299B8 opt, pGL4.299B8 opt-P or pGL4.29. Mixing plasmid DNA with 850. mu.L of OPTI-

(Invitrogen) and 32.4. mu.L of

HD transfection reagents (Promega Corp.) were mixed and incubated at room temperature for 10 minutes. Add 8 μ Ι _ of transfection/reporter DNA mixture to the appropriate wells (2 constructs/plate). Cells were incubated at 37 ℃ for 4 hours. By OPTI-

+ 0.5% dialyzed FBS +1X NEAA +1X sodium pyruvate +1X penicillin-streptomycin replacement medium and incubated overnight at 37 ℃.

After incubation, OPTI-containing 10nM or 10. mu.M FSK (from 10X stock solution)

Added to the wells and incubated at 37 ℃ for 3 hours. The mixture was washed with 100mM MES pH 6.1, 1mM CDTA, 150mM KCl, 35mM thiourea, 2mM DTT, 0.25%

NP-9(v/v)，0.025％

DF 204 and 20. mu.M of lysis reagent for PBI-3939 were added to cells containing pGL4.299B8 opt or pGL4.299B8 opt-P and incubated at room temperature for 10 min (100. mu.L of lysis reagent was added to 100. mu.L of cells). Subjecting ONE-GLO^TMAssay reagents (Promega Corp.) were added to pgl 4.29-containing cells and used according to the manufacturer's protocol (100 μ L reagent added to 100 μ L cells). In that

On the photometerThe light is emitted quantitatively. Table 26 shows the luminescence of HEK293 cells expressing transcriptional reporters containing CRE treated with 10nM ("baseline") or 10mM FSK, and the response to FSK (i.e., the luminescence generated by 10mM FSK treated cells divided by the luminescence generated by 10nM FSK treated cells).

The results shown in Table 28 indicate that 9B8 opt and 9B8 opt-P were brighter than luc2P and that all luciferase reporters responded to FSK when transfected with 100ng of DNA. However, when transfection was performed using only 10ng of DNA, the luminescence of luc2P was below the detection level of the luminometer.

Table 28: CRE-containing transcriptional reporter in HEK293 cells (3 hour time point)

Reverse transfection

Transcription reporters containing Antioxidant Response Element (ARE) and 9B8 opt or 9B8 opt-P were prepared in pGL4.29(Promega Corp.) backbone using protocols known in the art according to the manufacturer, i.e., the luc2P gene of pGL4.29 vector was replaced by 9B9 opt or 9B8 opt-P and CRE was replaced by 2 XARE (SEQ ID NO: 66).

HEK293 cells (T75 flask, 3mL pancreatin) were trypsinized and incubated at 1X10⁵One cell/mL (about 8.9X 10)⁶Total cells) were resuspended in medium containing DMEM + 10% FBS +1X NEAA. By mixing 1.2mL OPTI-

12 μ L of transcription reporter DNA (100 ng) and 36 μ L

HD transfection reagents were mixed together to prepare each transcription reporter for transfection and incubated for 35 minutes at room temperature. After incubation, 624 μ L of the transfection/reporter DNA mixture was added to the 12mL cell suspension and mixed by inversion. After mixing, 100 μ Ι _ of cell/DNA mixture was added to the wells of a 96-well plate (2 constructs/plate). Incubating at 37 deg.CAnd (5) carrying out cell for 22 hours. OPTI-containing formulations containing tert-butyl-p-benzenediol (Nrf2 stabilizer; tBHQ; 1. mu.M ("base line") or 20. mu.M) or sulforaphane (known as an organosulfur antioxidant activating Nrf 2; 1. mu.M ("base line") or 20. mu.M)

Added to each well and incubated at 37 ℃ for 24 hours. Cells were lysed using 100 μ L of lysis reagent as described above for forward transfection. In that

Luminescence was measured on a luminometer.

Table 29 shows the luminescence and response to sulforaphane for HEK293 cells expressing AREs containing transcriptional reporter treated with 1 μ M ("baseline") or 20 μ M sulforaphane (i.e., by dividing the luminescence generated by 1 μ M sulforaphane treated cells by the luminescence generated by 20 μ M sulforaphane treated cells). Table 30 shows the luminescence and response to tBHQ for HEK293 cells expressing the arecontaining transcriptional reporter treated with 1 μ M ("baseline") or 20 μ M tBHQ (i.e., by dividing the luminescence generated by 1 μ M tBHQ treated cells by the luminescence generated by 20 μ M tBHQ treated cells). Tables 29 and 30 show that 9B8 opt and 9B8 opt-P ARE able to report the presence of two different known ARE stimuli.

Table 29: ARE-containing transcriptional reporter in HEK293 cells (24 hour time points)

Table 30: ARE-containing transcriptional reporter in HEK293 cells (24 hour time points)

Bulk transfection

Transcriptional reporters containing CRE and 9B8 opt or 9B8 opt-P as described in the forward transfection were used in bulk transfection of HEK293 and NIH3T3 cells. Transcription reporters containing a heat shock response element (HRE; SEQ ID NO:67) and either 9B8 opt or 9B8 opt-P were prepared in pGL4.29(Promega Corp.) backbone, i.e., the luc2P gene of pGL4.29 was replaced with 9B9 opt or 9B8 opt-P and CRE was replaced with HRE. Transcriptional reporters containing HRE and 9B8 opt-P were used in bulk transfection of HeLa cells.

HEK293, NIH3T3 or HeLa cells were seeded into individual wells of a 6-well tissue culture plate the day before transfection at a density of 4.5X10 in 3mL complete medium (DMEM + 10% FBS +1X NEAA +1X sodium pyruvate) for HEK293 cells⁵Individual cells/well, 3X10 in 3mL complete medium (DMEM + 10% Fetal Calf Serum (FCS) +1X NEAA +1X sodium pyruvate) for NIH3T3 cells⁵Individual cells/well, or 9.9X10 in 3mL complete medium (DMEM + 10% FBS +1X NEAA) for HeLa cells⁵Individual cells/well. Cells were grown overnight at 37 ℃.

155 μ L of OPTI-

3,300ng of reporter plasmid DNA with 9.9. mu.L

HD transfection reagents were mixed, vortexed briefly, and incubated at room temperature for 10 minutes. HEK293 and NIH3T3 cells were transfected with CRE transcription reporter. HeLa cells were transfected with HRE transcription reporter. The reporter mix was added to the cells and mixed by gentle shaking, then incubated at 37 ℃ for 6 hours (HEK293 and NIH3T3) or 3 hours (HeLa). The cells were then trypsinized and resuspended in culture medium (DMEM + 10% FBS +1X NEAA +1X sodium pyruvate for HEK293 cells, DMEM + 10% FCS +1X NEAA +1X sodium pyruvate for NIH3T3 cells or DMEM + 10% FBS +1X NEAA for HeLa cells) and then plated into individual wells of a 96-well plate (20,000 cells/100. mu.L for HEK293, 10,000 cells/100. mu.L for NIH3T3μ L, or 13,000 cells/μ L for HeLa) and incubated overnight at 37 ℃.

OPTI-containing FSK (CRE stimulator) or 17-AAG (HRE stimulator; 17-allylamino-demethoxygeldanamycin)

Added to cells (10 nM or 10. mu.M final concentration for FSK; 1nM or 1. mu.M final concentration for 17-AAG) and incubated at 37 ℃ for 4 hours (FSK) or 6 hours (17-AAG). Remove the plate from the incubator and allow to equilibrate to room temperature for 25 minutes. Cells were lysed with 100 μ L lysis reagent as described above for forward transfection. In that

Luminescence was measured on a luminometer.

Table 31 shows the luminescence, and response to FSK, of HEK293 cells expressing transcriptional reporters containing CRE treated with 10nM ("baseline") or 10mM FSK. Table 32 shows the luminescence, and response to FSK, of NIH3T3 cells expressing transcriptional reporters containing CRE treated with 10nM ("baseline") or 10mM FSK. Table 33 shows the luminescence, and response to 17-AAG, of HeLa cells expressing transcriptional reporters containing HRE treated with 10nM ("baseline") or 10mM 17-AAG.

Tables 29-31 show that 1) in the context of two different cell lines HEK293 and NIH3T3, two forms of the 9B8opt OgLuc variant can report the presence of FSK and the stimulatory effect of FSK on CRE, and 2) in the context of HeLa cells, 9B8 optP can report the presence of 17-AAG and the stimulatory effect of 17-AAG on HRE.

Table 31: CRE-containing transcriptional reporter in HEK293 cells (4 hour time points)

Table 32: CRE-containing transcriptional reporter in NIH3T3 cells (4 hour time point)

Table 33: HRE-containing transcriptional reporter in HeLa cells (6 hour time points)

Example 38 lytic and secreted reporter in difficult to express cells

According to manufacturer's instructions

HD uses plasmid DNA (pGL4.32 backbone, Promega Corp.) containing L27V02, luc2P (Promega Corp.), luc2(Promega Corp.) or L27V02-IL6 (L27V 02 containing a native secretory sequence replaced by an IL-6 secretory sequence; IL601-L27V 02A; SEQ ID NO:324) against 1x10 in cell suspension ⁵HepG2 cells were transfected in reverse direction (1:20 DNA-transfection mixture to cells) per mL of cells. Then 100. mu.L of the cell suspension was seeded into wells of a 96-well plate and incubated at 37 ℃ with 5% CO₂Incubate for 22 hours. Other OgLuc constructs with native secretory sequences replaced by IL-6 secretory sequences include IL601-L27V01 and IL602-L27V03 (SEQ ID NOS: 321 and 327, respectively).

For secretion analysis, the medium was removed from the cells and the cells were washed in 100 μ L DPBS. 100 μ L of complete medium (DMEM + 10% FBS +1X NEAA) and various doses (1pg/mL-100ng/mL) of rhTNF α ("TNF α") were added for 4.5 hours. 10 μ L of the medium was removed, 90 μ L of complete medium was added thereto, and 100 μ L of assay reagents (as described previously; 100 μ M PBI-3939) were added. Luminescence was measured as previously described (fig. 62A).

For lysis assay, cells were seeded at 37 ℃ with 5% CO₂Incubate for 4.5 hours. The cells were then allowed to equilibrate to room temperature for 20 minutes. Assay reagents (as described previously; 100. mu.M PBI-3939) were added to the cells and luminescence was measured as described previously (FIG. 62B).

Example 39 additional cleavage reporter features

In a cell-based environment, the OgLuc variants of the invention, the cleaved transcription reporter should provide a luminescent signal of an intensity such that the signal appears faster than it might appear with other luciferase enzymes. This bright luminescence also allows the examination of weak promoters.

Example 40 mammalian cell transfection

The OgLuc variants of the invention are useful as reporters in difficult to transfect cell lines, e.g., Jurkat, HepG2, primary cells, non-dividing primary cells or stem cells (see, fig. 59C). Due to its high signal intensity, the OgLuc variant is able to make luminescence detectable when transfection efficiency is low. The OgLuc variants can also be used as a reporter in cells that are sensitive to, among other things, conditions associated with transfection (i.e., DNA concentration, addition of transfection reagents). Due to the brightness of the OgLuc variant, adequate levels of luminescence can be obtained with lower DNA concentrations, less transfection reagent, and possibly shorter post-transfection time before the start of the assay. This will make the sensitive cells less likely to be subjected to toxic loads. The bright luminescence of the OgLuc variant should also allow the detection of signals at very long time points, in events such as those requiring output. As another example, the OgLuc variant may use a reporter that acts on a single copy of a native promoter, such as the HSB Thymidylate Kinase (TK) promoter, the HOX gene, or LIN 28.

Example 41 Stable cell lines

The bright signal of luciferase and the small size of the OgLuc gene can facilitate the identification of robust, stable cell lines that express the cytoplasmic or secreted forms of the OgLuc variants of the invention. This relatively small gene sequence should reduce the possibility of gene instability due to integration of foreign DNA.

To generate stable cell lines using the OgLuc variants of the invention, plasmid DNA comprising the nucleotide sequences of the OgLuc variants and a selectable marker gene (e.g., neomycin, hygromycin, or puromycin) is used to transfect a cell line of interest, e.g., HEK293 cells. Cells of early passage numbers, e.g., less than 10 passages, are seeded into T25(1X 10)⁶) Or T75(3x10⁶) Tissue culture flasks and allowed to grow overnight to about 75% confluence. The plasmid DNA and appropriate transfection reagents are then used, for example,

-LT1 or

HD to transfect cells. 48 hours after transfection, the cell culture medium was replaced with selection medium containing a selection drug such as G418, hygromycin or puromycin at a concentration previously determined to kill untransfected cells. Cells containing plasmid DNA were selected after 2-4 weeks. During this time, cells were re-seeded at different concentrations into selection media of T25 or T75 tissue culture flasks. The medium on the reseeded cells was replaced with fresh selection medium every 3-4 days for 2-3 weeks. Flasks were monitored for the formation of viable cell clones. Eventually, the flask will contain many large clones and few dead cells.

From the mixture of stable clones in flasks, single clones were isolated and expanded into individual 24-well tissue culture plates. Briefly, cells were harvested using the trypsin/EDTA method, i.e., by removing the medium, with Ca-free media²⁺And Mg²⁺The cells were harvested by rinsing with PBS and detached by treatment with pancreatin/EDTA. Count cells using a hemocytometer and count 1 × 10⁵Diluted in complete medium. The cells were then diluted in complete medium to 100 cells/mL, 33 cells/mL, 10 cells/mL and 3.3 cells/mL. 100 μ L of each dilution was inoculated into all wells of a 96 well tissue culture plate (1 plate per dilution) and allowed to grow for 4-5 days before 50 μ L of selection medium was added to the cells. Approximately 1 week after inoculation, clones of the screened cells were grown visually and another 50. mu.L of selection medium was added. Cells were monitored continuously until the single clone covered 40-60% of the well area. When the clones are to be amplified and screened, the clones are harvested using the pancreatin/EDTA method. Each clone was transferred to selection medium as follows: 1)1:10 dilution into 6 wells of a 96-well assay plate for functional assays (e.g., luminescence detection); 2)1:10 to For cell viability assays (e.g., CELLTITER-

Luminescent Cell Viability Assay (Promega Corp.) in 3 wells of a 96-well clear-bottomed Assay plate; 3)1:10 dilution into 24-well tissue culture plates for amplification. The cells in the plates used for the functional and cell viability assays were then cultured for 2-3 days and subjected to the functional and cell viability assays. Positive clones in 24-well plates were then further tested using functional assays and cell viability assays and tested for stability and response of expression for at least 20 passages, normal growth rate morphology, and frozen at the earliest possible passage for future use.

Example 42 OgLuc secretion Signal analysis

A.IV opt

The wild-type OgLuc was processed after synthesis into a mature protein with the secretion signal sequence cleaved off. To determine whether this secretion signal sequence will aid in secretion of the OgLuc variant, the IV opt variant of example 25 and hRL were cloned into pF4Ag containing the N-terminal OgLuc secretion signal (SEQ ID NO: 54). HEK293 cells (15,000) in 100. mu.L of Darber's modified eagle's medium ("DMEM") containing 10% Fetal Bovine Serum (FBS) were transfected as described in example 25 using 100ng of plasmid DNA, i.e., hRL or IV opt with or without secretion signal and grown overnight at 37 ℃. 50 μ L of medium was removed to a new plate and stored for later assay, generating a "medium" sample. The remaining medium was removed and the cells were lysed with 100 μ L of lysis buffer as described in example 25 to generate a "lysate" sample. Luminescence was measured for 10. mu.L of the culture medium samples and 10. mu.L of the lysate samples (FIG. 63). Samples of hRL with or without the OgLuc secretion signal sequence ("Renilla sig") were measured using 50. mu.L of lysis buffer containing 20. mu.M native coelenterazine. Samples of IV with or without the OgLuc secretion signal sequence ("IV opt sig") were measured using 50. mu.L of lysis buffer containing 20. mu.M PBI-3939.

In fig. 63, the solid bars represent the amount of light detected from the medium in the absence of any lysis reagent. Open bars represent total light detected after addition of lysis reagent (secreted + non-secreted). Figure 63 shows IV opt secreted from HEK293 cells into growth medium and that the secretory signal sequence is functional in mammalian cells. "IV opt sig" represents the only case where a significant amount of luciferase was detected in the medium. The results also indicate that this particular signal peptide does not assist in the secretion of hRL.

B.9B8, V2 and L27V

To determine whether the secretion signal sequence of OgLuc helped its secretion, the OgLuc variant 9B8, V2, and L27V were cloned into pF4Ag containing the N-terminal OgLuc secretion signal sequence. The variants were also cloned into a vector without a secretion signal sequence. CHO or HeLa cells were then seeded at 100,000 cells/well into 12-well plates in 1mL of F12 medium (CHO cells) containing 10% FBS and 1X sodium pyruvate or DMEM (HeLa cells) containing 10% FBS and 1X sodium pyruvate at 37 ℃ with 5% CO₂Incubate overnight.

After overnight incubation, use was made of

LT1 transfection reagent (Mirus Bio) and OPTI-

Media (Invitrogen) cells were transfected with 1. mu.g of DNA containing 9B8, V2 or L27V with or without secretion signal sequences. The cells were again incubated at 37 ℃ with 5% CO ₂Incubate overnight.

After the second overnight incubation, the medium was removed and stored for analysis. To the cells was added 1mL of assay buffer (1mM CDTA, 150mM KCl, 2mM DTT, 100mM MES pH 6.0, 35mM thiourea and 0.5%

NP-9(v/v)) to produce a cell lysate. To 10. mu.L of cell lysate or stored media from each sample was added 50. mu.L of assay buffer containing 40. mu.M PBI-3939Liquid and the luminescence was measured as described above. FIGS. 64A-D show that the 9B8, V2, and L27V variants can be used in secretable systems.

To determine the stability of secreted variants, 150 μ L aliquots of the stored media from each sample were placed at 37 ℃ or 50 ℃. Aliquots were then removed at different time points (0, 1, 2, 3, 5, 6 and 7 minutes), frozen on dry ice, and stored at-20 ℃ until assayed. To determine stability, aliquots of the medium were thawed to room temperature and 10 μ L of each aliquot was mixed with assay buffer containing PBI-3939(pH 6.0) as described above. Luminescence was measured as above and the half-life (t) determined₅₀) (Table 34).

Watch 34

Sample (I)	Half life 37 ℃ (days)
		9B8	8
V2	10
		L27V	17
		Sample (I)	Half-life 50 ℃ (hour)
9B8	3
		V2	7
L27V	11

C.9B8 and V2 comparison of secreted luciferases from Metridia elongata

OgLuc variants 9B8 and V2 were compared with respect to secretion of secreted luciferase from daphnia elongata. CHO cells were seeded at 300,000 cells/well in 3mL F12 medium containing 10% FBS in wells of a 6-well plate and 5% CO at 37 ℃%₂The cells were incubated overnight. And then utilized according to the manufacturer's instructions

LTI cells transfected with 10 or 100ng of each variant or Metridia luciferase (Clontech) plasmid DNA and 5% CO at 37 ℃₂Incubate for 20 hours. After transfection, the medium was removed from the cells and assayed. For the OgLuc variant, 50. mu.L of medium was assayed using 50. mu.L of assay reagent (previously described; 40. mu.M PBI-3939). For Metridia luciferase, Ready-to-Glo was utilized according to the manufacturer's protocol^TMThe culture medium was assayed by the selected Luciferase Reporter System (Clontech). Briefly, 5. mu.L of 1 Xsubstrate/reaction buffer was added to 50. mu.L of the culture medium sample. Luminescence was then measured as previously described (fig. 65A-B).

Example 43 evaluation of OgLuc variants and novel coelenterazine in Living cells

A. The use of the OgLuc variant and PBI-3939 in living cells was examined. HEK293 cells were seeded at 15,000 cells/well in 96-well plates and grown overnight at 37 ℃. The following day, 100ng of pF4Ag containing either hRL or 9B8 opt was used for three recycles

LT1 was transiently transfected into cells and grown overnight at 37 ℃. The following day the growth medium was removed and used with a medium containing 60. mu.M VIVIVIREN^TM Live Cell Substrate(Promega Corp.)，60μM ENDUREN^TMLive Cell Substrate (Promega Corp.) or medium replacement for 60 μ M PBI-3939 for both hRL and 9B8 opt transfected cells. Untransfected cells were used as background controls. Incubate the plate at 37 ℃ during the course of one day and incubate at

GENIOS^TMMeasurements were performed periodically on the Pro photometer, i.e. 11 times during 24 hours. FIGS. 66A-B show the luminescence of transfected cells divided by the luminescence of untransfected cells for each substrate, i.e., the ratio of signal to background. Data show the result of using VIVIVIN^TM，ENDUREN^TMOr PBI-3939 in a background of viable cells (i.e., not lysed) of 9B8 opt. The data also indicate that PBI-3939 is able to penetrate cells in culture, react with the OgLuc variant and generate luminescence, making it compatible with use in live cell assays.

B. To demonstrate viable cell analysis using the OgLuc variant, L27V was compared with

Fusion and expression in living cells and monitoring. U2OS cells were seeded into imaging chamber wells at 40,000 cells/mL and 5% CO at 37 ℃₂Incubation was performed overnight. Then using plasmids pFC14K, pFN21K or pF4Ag (all Promega Corp.) containing L27V or pF4Ag containing L27V with natural or IL-6 secretion sequence according to the manufacturer's protocol

HD transfected cells. Then 5% CO at 37 ℃₂Cells were incubated for 24 hours.

After incubation, the cells are incubated with

TMR (Total Mixed Ration) assemblyThe bodies (Promega Corp.) were contacted, imaged and fixed. Then according to

ICC scheme in the art: a focused imaging technical Manual (Promega Corp.; TM260) was used for Immunocytochemistry (ICC). The primary antibody used was polyclonal rabbit, anti-OgLuc 9B8 antibody (1: 1000). The secondary antibody used was Alexa 488 (green) conjugated to the secondary antibody (fig. 67A). FIG. 67A shows the green fluorescence channel and FIG. 67B shows the Differential Interference Contrast (DIC). Using a gas turbine equipped with 37 ℃ + CO₂Images were acquired with an Olympus Fluoview FV500 confocal microscope (Olympus, USA) from an environmental chamber (solvent Scientific ltd., UK).

FIGS. 67B-D show ICC images with native or IL-6 secretory sequences. Both signal sequences significantly increased the amount of enzyme in the nucleus. The highlighted nature of the marker in the cytoplasm represents the vesicle formation that is expected to occur during secretion. The data indicate that the presence of the signal peptide reduces the amount of luciferase in the nucleus.

C. As indicated above, the OgLuc variants and novel substrates of the invention are biocompatible. Reporter systems are envisioned in which the OgLuc variant is cloned into an expression vector containing the promoter of interest and expressed in the cell as a reporter protein. The cells were then treated with PBI-3939 which would permeate the cells in culture, react with the OgLuc variants, and generate luminescence.

In addition to being a cell permeate, PBI-3939 showed biocompatibility comparable to native coelenterazine in terms of cell viability. One form of compound 3939 containing chemical modifications known to increase the stability of native coelenterazine in culture media can be synthesized and used for robust, living cell OgLuc variant-based reporter assays. Another example of a live cell report includes the use of a secretable OgLuc variant as a reporter. A native secretion signal peptide (or other known secretion signal peptide) can be fused to the N-terminus of the OgLuc variant so that when the fusion is expressed in a lactating animal, part of it will be secreted into the culture medium through the cell membrane. Luminescence is generated upon addition of the substrate.

Example 44 protein fusion reporter

The OgLuc variants of the invention can be used as fusion tags for a target protein of interest as a means of monitoring intracellular levels of the target protein. Specific proteins involved in stress response pathways, e.g., DNA damage, oxidative stress, inflammation, can be monitored in cells as a means of exploring the different items of stimuli that may play a role in these pathways. The variants can also be used as a means of monitoring cellular trafficking of target proteins. Variants can also be fused to viral genomes (e.g., HIV, HCV) so that titer levels, i.e., infectivity, can be monitored in cells after treatment with potential antiviral agents. Variants may also be associated with Green Fluorescent Protein (GFP) or

Fusion (in addition to the target protein) so that FACS can be used to identify high expressing clones and provide localization information.

Example 45-evaluation of OgLuc variants in 3-Module fusion proteins ("sandwiches")

3-component fusion proteins or "sandwich" fusions that can be used to place bioluminescent and fluorescent proteins in close proximity to each other to optimize BRET-based biological sensors.

C1+4AE, IV, 9B8 and 9F6

The OgLuc variants C1+4AE (SEQ ID NOS: 55 and 56), IV (SEQ ID NOS: 57 and 58), 9B8(SEQ ID NOS: 61 and 62) and 9F6(SEQ ID NOS: 63 and 64) and hRL (SEQ ID NOS: 32 and 33) were cloned into pF4Ag fusion vectors using an N-terminal Id (Benezra et al, Cell, 61(1):49-59(1990)) and a C-terminal HT7 for normalization, which are known poor fusion partners. The gene of interest was "sandwiched" between Id and HT7 (i.e., Id-luciferase-HT 7), e.coli lysates were prepared as described in example 26 containing either pF4Ag or the variant construct in pF4Ag sandwich background, and then assayed with 20 μ M native coelenterazine in buffer as described in example 25.

Fig. 68 shows the luminescence of each variant in a pF4Ag or pF4Ag background ("sandwich"). Figure 69 shows the fold increase in luminescence due to Id and HT7, and was determined by dividing the luminescence of the variant in pF4Ag by the luminescence of the variant in pF4 Ag-sandwich. The sample with the maximum value showed the strongest sensitivity to the poorer fusion partner Id. Variant 9B8 was the brightest in the sandwich environment.

B.9B8 OPT and 9B8 OPT + K33N

Variants 9B8 opt and 9B8 opt + K33N were analyzed in a sandwich background as previously described. Sandwich constructs of 9B8 opt (SEQ ID NOS: 40 and 41) and 9B8 opt + K33N (SEQ ID NOS: 59 and 60) were generated as previously described. Coli lysates were assayed and measured using the same assay buffer and luminometer as used to generate figure 40. FIG. 70 shows the fold increase in the presence of the sandwich background, indicating that 9B8 opt + K33N is less sensitive to poor fusion partner Id than 9B8 opt.

23D2 and 24C2

Variants 23D4(NF) and 24C2(NF) were subcloned into the Id-OgLuc-HT7 sandwich background and assayed in E.coli. Sandwich variants 23D4(F) (SEQ ID NOS: 76 and 77) and 24C2(F) (SEQ ID NOS: 78 and 79) were compared to 9B8 opt + K33N (SEQ ID NOS: 59 and 60) in a sandwich background. Table 35 shows that the variants have at least the same luminescence as 9B8 opt + K33N in the sandwich background environment.

Table 35: increase in luminescence generated by OgLuc variants compared to 9B8 opt + K33N +170G in a Sandwich background

D.1F7 and 15H1

Screening for additional variants of the PCR library with increased luminescence in the Id-OgLuc-HT7 sandwich background compared to 9B8 opt + K33N in the sandwich background. The selected variants were then assayed in HEK293 cells and NIH3T3 cells. Table 36 shows the fold increase in luminescence of the sandwich variants and the amino acid substitutions found in the variants in enterobacter coli cells, HEK293 cells and NIH3T3 cells. 1F7(F) (SEQ ID NOS: 84 and 85) and 15H1(F) (SEQ ID NOS: 86 and 87) had at least a 1.3-fold increase in luminescence in E.coli. 1F7(F) was brighter in HEK293 cells and NIH3T3 cells than 9B8 opt + K33N in the sandwich background.

Table 36: increase in luminescence generated by OgLuc variants compared to 9B8 opt + K33N in a Sandwich background

The sandwich variants were cloned into a non-fusion background vector based on pF4Ag to generate 1F7(NF) (SEQ ID NOS: 80 and 81) and 15H1(NF) (SEQ ID NOS: 82 and 83) and analyzed as previously described and compared to 9B8 opt + K33N. Table 37 shows the fold increase in luminescence of the variants in e.coli cells, HEK293 cells and NIH3T3 cells. 1F7(NF) and 15H1(F) had at least a 1.3 fold increase in luminescence in E.coli and HEK293 cells.

Table 37: increase in luminescence generated by OgLuc variants compared to 9B8 opt + K33N +170G in a Sandwich background

V2, 9B8 opt + K33N + L27V + K43R + Y68D, 9B8 opt + K33N + L27V + T39T + K43R + S66N and L27V

The variants 9B8 opt + K33N + T39T + K43R + Y68D ("V2"; SEQ ID NOS: 92 and 93), 9B8 opt + K33N + L27V + K43R + Y68D (SEQ ID NOS: 339 and 340), 9B8 opt + K33N + L27V + T39T + K43R + S66N (SEQ ID NOS: 341 and 342) and 9B8 opt + K33N + L27V + T39T + K43R + Y68D ("L27V"; SEQ ID NOS: 88 and 89) were subcloned into the Id-OgLuc-HT7 sandwich background and assayed in HEK293 and NIH3T3 cells as previously described. The luminescence generated by the sandwich variant was compared to the luminescence generated by the 9B9 opt + K33N sandwich (SEQ ID NOS: 59 and 60) (Table 38). The L27V sandwiches (SEQ ID NOS: 90 and 91) and the V2 sandwiches (SEQ ID NOS: 94 and 95) had at least a 1.3X fold increase in luminescence in HEK293 cells and NIH3T3 cells.

Table 38: increase in luminescence generated by OgLuc variants in the Sandwich background compared to 9B8 opt + K33N in the Sandwich background

Sample (I)	NIH3T3 cells	HEK	293
				K33N Sand	1.0	1.0
T39T，K43R，Y68D Sand	1.6	2.3
			L27V，K43R，Y68D Sand	1.4	1.7
L27V，T39T，K43R，S66N Sand	0.7	0.7
			L27V，T39T，K43R，Y68D Sand	1.4	1.7

Sandwich and non-sandwich forms of variants V2, 9B8 opt + K33N + L27V + K43R + Y68D, 9B8 opt + K33N + L27V + T39T + K43R + S66N and L27V were determined in HEK293 cells and NIH3T3 cells as described in example 37. The luminescence generated by the non-sandwich variant was compared to the luminescence generated by the sandwich variant (table 39). The data shown in table 39 indicate that the fold reduction in luminescence for the 9B8 opt + K33N sandwich was lower in mammalian cells than in e.coli cells, as shown in figure 70.

Table 39: fold reduction of luminescence of OgLuc variants in the Presence of Sandwich background

Sample (I)	NIH3T3 cells	HEK	293
				K33N	29	15
T39T，K43R，Y68D	20	6
			L27V，K43R，Y68D	22	8
L27V，T39T，K43R，S66N	25	12
			L27V，T39T，K43R，Y68D	18	6

Example 46 multiple compounding

A. Lysates of E.coli expressing variant 9B8 opt were prepared as previously described in example 27 and were purified in the absence of phenol Red + 0.1%

Diluted 1000-fold in DMEM. Using modified DUAL-

The Luciferase Assay System (Promega Corp.) measures luminescence from a sample containing 6.3. mu.g/mL of purified red click beetle Luciferase and E.coli lysate expressing the variant 9B8 opt. According to the manufacturer's protocol, use of DUAL-

STOP&

Reagent and DUAL-

STOP&

Reagent to detect red click beetle luciferase and OgLuc variant 9B8 luciferase from a single sample. Three replicates were performed.

In Turner MODULUS^TMLuminescence was detected on a luminometer. Table 40 shows the average luminescence generated by red click beetle luciferase ("click beetle"), and the luminescence generated by 9B8 opt ("OgLuc") using coelenterazine-h ("coelenterazine h") or PBI-3939 ("3939"). The standard deviation ("+/-") and the coefficient of variation ("CV") are also shown. A "No coelenterazine" control was performed to show the results obtained by DUAL-

DUAL-

STOP&

Amount of red click beetle signal quenched by Reagent. The "no coelenterazine" control produced 349-fold quenching. Table 40 shows large luminescent signals from both red click beetles and the OgLuc variant 9B8 detected in a single sample. This indicates that each signal can be read out sequentially in a two step assay and that the signal from the first enzyme can be quenched sufficiently without significantly affecting the signal from the second enzyme.

Table 40: using modified DUAL-LUCIFERASE^TMReporter assay mean luminescence generated by Red click beetles and 9B8 opt luciferase

B. To demonstrate that the multiplex reporter assay described above can be run in reverse, i.e., first detecting OgLuc luminescence, quenching and detecting a second luminescence, e.g., red click beetles or luciferase, various renilla luciferase inhibitors (see U.S. published application No. 2008/0248511) were screened for the ability to also inhibit OgLuc. Two different previously identified Renilla inhibitors PBI-3077 and 1424 were added at different concentrations (see Table 41) to E.coli lysate samples expressing variant 9B8 (diluted as above) and containing 100mM MES pH 6.0, 1mM CDTA, 150mM KCl, 35mM thiourea, 2mM DTT, 0.25%

NP-9(v/v)，0.025％

DF 204 and 20. mu.M PBI-3939 in buffer. Measuring luminescence as described previously, except using

Multi micro-plate photometer (Promega Corp.; also known as Turner MODULUS)^TM) Luminescence was measured. As shown in table 41, both compounds were able to inhibit OgLuc luminescence. This indicates that the OgLuc variant can be multiplexed with another luciferase in a reporter assay in which the luminescence from the OgLuc variant is first detected.

Table 41: effect of PBI-3077 and PBI-1424 on luminescence generated from bacterial lysates expressing 9B8 opt using PBI-3939 as substrate

	(mM or%)	RLU	+/-	％
						Control	27,794,600	626,862	100％
Al(3077)	-	15,473,100	209,567	56％
					mM	0.3	22,210,433	102,888	80％
	0.03	22,484,933	927,459	81％
					AC(1424)	0.4	176,868	9,579	0.64％
％	0.04	24,267,533	363,861	87％
						0.004	25,126,900	1,569,453	90％

C. Spectral resolution was analyzed between the OgLuc variant L27V and the firefly luciferase (Fluc). Will not contain phenol red + 0.1%

Purified L27V in DMEM (previously described; 9.54pM) was mixed with assay reagents (previously described) containing 20. mu.M PBI-3939. Mixing purified firefly luciferase (f) in the same medium

A recombinant luciferase; promega corp.; 271ng/mL) with detection reagent (100mM HEPES, pH 7.4, 1mM CDTA, 16mM MgSO4, 1%

NP-9(v/v)，0.1％

DF 204, 5mM ATP, 50mM DTT, 333. mu.M fluorescein). Purified Renilla Luciferase (5ng/mL GST-Renilla) from 1X Renilla Luciferase Assay lysine Buffer (Promega Corp.) was mixed with 10.5. mu.M native coelenterazine in Renilla Luciferase Assay Buffer. Luminescence of L27V and Fluc was measured after 3 minutes and luminescence of renilla luciferase was measured after 10 minutes (fig. 71).

D. As another example, the OgLuc variants of the invention can be used as transcription reporters and paired with (or simultaneously with) firefly luciferase biosensors that exchange with aequorin or cAMP cycle to detect multiple pathways in a single sample, e.g., aequorin for detecting and/or measuring calcium, biosensors for detecting and/or measuring cAMP, and OgLuc variants for monitoring downstream gene expression.

E. Other examples of multiple complexation with the OgLuc variants of the invention include:

i) cells are transfected with constructs comprising an OgLuc variant of the invention and a firefly luciferase. Following transfection, a first reagent may be added to lyse the cells and provide a substrate to generate luminescence for the first luciferase. Luminescence from the first luciferase can then be measured. A second reagent may then be added to quench the luminescence from the first luciferase and provide a substrate to generate luminescence from the second luciferase. Luminescence from the second luciferase can then be measured. The choice of which luciferase to measure first will depend only on the ability to quench the luminescence from the first luciferase with the second reagent. For this example, luminescence from the OgLuc variant can be first measured, since luciferin (a substrate for firefly luciferase) has been shown to inhibit the activity of the OgLuc variant at high concentrations.

ii) transfecting the cell with a construct comprising an OgLuc variant of the invention and a firefly luciferase. Following transfection, a first reagent may be added which contains a living cell substrate to generate luminescence for the first luciferase. The luminescence from the first luciferase will then be measured. A second reagent is then added to lyse the cells, quench the luminescence from the first luciferase and provide a substrate to generate luminescence from the second luciferase. The luminescence from the second luciferase will then be measured. This is similar to i), except that cell lysis will further limit the use of viable cell substrate and facilitate quenching of luminescence from the first luciferase.

iii) transfecting cells with the OgLuc variant of the invention and a construct of firefly luciferase. After transfection, a reagent may be added which contains a substrate to generate luminescence from both luciferases, but the luminescence from each luciferase is spectrally different. The OgLuc variant has an emission maximum of about 460nm and certain substrates of firefly luciferase, e.g., 5 '-chloroluciferin and 5' -methylluciferin, can produce an emission maximum of about 610 nm. Thus, there may be some overlap from blue emission to red emission and no overlap from red emission to blue emission, indicating that little or no mathematical correction may be involved.

iv) transfecting the cell with a construct comprising an OgLuc variant of the invention and a firefly luciferase. After transfection, an agent may be added which contains a living cell substrate to generate luminescence from both luciferase enzymes. The unique feature of this example is that firefly luminescence tends to shift to red at live cell assay temperatures, e.g., 37 ℃, and thus, a large number of different luciferin derivatives can be selected as the live cell substrate for firefly luciferase to produce luminescence that is spectrally distinct from the OgLuc variant.

v) transfecting the cell with a construct comprising an OgLuc variant of the invention and a Renilla luciferase. Following transfection, a first reagent may be added to lyse the cells and provide a substrate to generate luminescence for the first luciferase. The luminescence from the first luciferase will then be measured. A second reagent will then be added to quench the luminescence from the first luciferase and provide a substrate to generate luminescence from the second luciferase. The luminescence from the second luciferase will then be measured. The choice of which luciferase to measure first depends only on the ability to quench the luminescence from the first luciferase with the second reagent. For this example, it is desirable to use an inhibitor that quenches luminescence of the OgLuc variant or renilla luciferase.

vi) transfecting cells with a construct comprising an OgLuc variant of the invention and a click beetle luciferase. After transfection, a reagent may be added which contains a substrate to generate luminescence from both luciferases, but the luminescence from each luciferase is spectrally different because the click beetle luciferases produce red-shifted luminescence using native luciferin.

Example 47 Cyclic exchange

Two cyclic exchange (CP) forms of the L27V variant were generated: CP84 and CP 95. Numerical designations refer to the N-terminal residues (e.g., "84" denotes the new N-terminus of the CP form).

To construct the circular crossover, the previous N-and C-termini were fused together between the N-and C-termini using NO linker ("CP 84 NO linker" (SEQ ID NOS: 97 and 98) and "CP 95 NO linker" (SEQ ID NOS: 105 and 106)) or 5 ("CP 845 AA linker" (SEQ ID NOS: 99 and 100) and "CP 955 AA linker" (SEQ ID NOS: 107 and 108), 10 ("CP 8410 AA linker" (SEQ ID NOS: 101 and 102) and "CP 9510 AA linker" (SEQ ID NOS: 109 and 110) or 20 ("CP 8420 AA linker" (SEQ ID NOS: 103 and 104) and "CP 9520 AA linker" (SEQ ID NOS: 111 and 112) an amino acid linker, (GSSGG) N (SEQ ID NO:113) to fuse the N-and C-termini together (note: L27V starts with phenylalanine from the N-terminus, i.e., MVF. "MV" is present in the "NO linker" construct, but not present in the "linker" construct). After circular crossover, the CP L27V variant was cloned into pF1KIn a carrier. Coli cells were transformed with the CP vector and cultured in minimal medium using the standard walk-through induction protocol described previously. For each CP construct, cells were grown in 8 wells of a 96-well plate. After induction, 8 wells from each sample were mixed and 10. mu.L lysed in 40. mu.L lysis buffer (100mM MES pH 6.0, 0.3X PLB, 0.3 mg/mL lysozyme, 0.003U/. mu.L DNase I and 0.25%

NP-9 (v/v)). The lysate was then diluted in lysis buffer at 1:100 (CP form with linker) or 1:1000 (CP-free form). The linker-free CP form was not diluted. The lysate or lysate dilution (previously described) was assayed in triplicate in 50 μ L of assay reagent. Luminescence was measured as previously described (fig. 72).

Example 48 identification of additional sites for circular crossover

To identify additional CP sites, determine the effect of CP sites on luciferase activity and investigate the use of "tethers" between fragments, CP constructs with circular exchanges at about every third site (i.e., amino acid) of the L27V variant were generated (see fig. 73E). One skilled in the art will appreciate that other sites, e.g., a first site and a second site, can also be examined and used in the cyclically-swapped OgLuc variants described herein using the protocol described herein according to the manufacturer.For example, it has been found that the L27V variant is particularly permissive for circulating exchange, particularly in the fragment of the exchange (e.g., based on cAMP @ Receptors for RIIbB) are placed between the binding domains.At each site, the linker GSSGG-GSSGG-EPTT-ENLYFQSDN-GSSGG-GSSGG (SEQ ID NO: 328). The underlined sequence refers to the TEV protease recognition site. The purpose of the linker is to provide a long enough tether between the two variant fragments that it can be ligated in a manner that results in a functional luciferase. The importance of maintaining activity can be investigated using the TEV protease recognition site to provide a means of disrupting the tether (in the presence of TEV protease). Use of TEV protease recognition sites A model was constructed to predict which Individual CP sites would be useful for Protein Complementation Assays (PCA) or for biological sensor applications (e.g., insertion of response elements between CP sites).

The activity seen before TEV cleavage represents how the two halves of the variant enzyme behave in the tethered state. Binding of TEV to the recognition site results in cleavage, separating the two halves of the variant enzyme. Samples that have lysed using TEV will represent an uninduced state and provide an indication of how much background can be expected. Lower activity after TEV cleavage indicates that the two halves cannot be linked together without induction. Samples that show a substantial loss of activity after TEV cleavage represent sites that will function in PCA and biosensor applications. In the case of PCA, the two halves of the variant enzyme will fuse with a binding partner capable of linking (tethering) after the induced binding event. In the case of biological sensors, the two halves will be "tethered" after the binding-induced conformational change occurs. An example of PCA would be to fuse half of L27V to FRB and the other half to FKBP. Two halves will be brought together upon exposure to rapamycin (Banaszynski et al, J.Am. chem.Soc, 127(13): 4715-. One example of a biological sensor application would be to insert a cyclic AMP binding domain (e.g., RIIbB) between CP sites and induce a conformational change by binding of the cyclic AMP to the binding domain.

After each CP L27V construct was generated, CP enzyme was expressed in wheat germ, e.coli and mammalian cells and digested with TEV protease to study luciferase activity.

1. For the analysis in wheat germs, use was made of the manufacturer's instructions

T7 Coupled Wheat Germ Extract System (Promega Corp.) expresses the CP construct. Will be provided with

The reaction was diluted 1:100 in 1 XPBS + 0.1% gelatin and 20. mu.L was added to 25. mu.L of TEV reaction reagent (5. mu.L of 20 XPProTEV buffer (Promega Corp.), 1. mu.L of 100mM DTT and 2. mu.L of 10U ProTEVPlus (Promega Corp.)). The volume of the digestion reaction was increased to 100. mu.L with water and incubated at 30 ℃ for 60 minutes. A control sample without TEV protease was also prepared. Then 10. mu.L of the digested sample was added to 40. mu.l DMEM to a final volume of 50. mu.L and assayed in 50. mu.L of assay reagent (as described previously; 100. mu.M PBI-3939). Luminescence was measured as previously described (fig. 73A-D).

2. For analysis in mammalian cells, HEK293 cells were transfected with the CP L27V variant using a reverse transfection protocol. Briefly, 1ng of CP L27V plasmid DNA was mixed with 1. mu.g of vector DNA and added to the cells in the wells of a 12-well plate. The cells were then incubated at 37 ℃ with 5% CO ₂Incubate for 16 hours. Cell lysates were then prepared by removing the culture medium from the cells, washing the cells in 1X PBS and adding 1mL of 1X PLB. The lysate was then diluted 1:10 in 1X PBS containing 0.1% gelatin. Then 40 μ L of the diluted lysate was used in TEV protease digestion as previously described. mu.L of digestion was mixed with 40. mu.L of DMEM without phenol red and 50. mu.L of assay reagent (previously described; 100. mu.M PBI-3939) was added. Luminescence was measured as previously described (fig. 73H).

3. For analysis in E.coli, E.coli cultures expressing the CP L27V variant were grown overnight at 30 ℃. These cultures (1:100 dilution in LB + antibiotics) were used to generate new starter cultures for final induction. The starter cultures were incubated at 37 ℃ for 2.5 hours (OD) with shaking₆₀₀About 0.5). Rhamnose (0.2% final concentration) was added, the culture was transferred to 25 ℃ and incubated for 18 hours with shaking.

To generate lysates, 50. mu.L of 0.5X FASTBREAK was added^TMCell lysis reagent (Promega Corp.) was added to 950 μ L of induced culture and the mixture was incubated at 22 ℃ for 30 minutes. 50 μ L of the lysed culture were then digested with TEV protease as previously described and incubated at room temperature for 2 hours.

For analysis, the lysate was diluted 1:10,000 in

In a Mammalian Purification Buffer (Promega Corp.) and 50. mu.L were assayed in 50. mu.L of assay reagents (as described previously; 100. mu.M PBI-3939). Substrate and TEV-induced luminescence were measured at 5 minute time points (fig. 73F) and responses were determined as previously described (fig. 73G).

FIGS. 73A-D show the basal luminescence of different CP-TEV protease L27V constructs expressed in wheat germ extract. Figure 73E summarizes the derived CP variants responding to TEV protease (response is fold-reduction), indicating that CP variants can be used as TEV receptors, i.e., they may represent "tether-dependence". The significance of variants showing at least a 1.2 fold change was further verified using student tests (unpaired p-values; confidence level 0.03). These results indicate that each CP variant is capable of generating luminescence.

Different CP-TEV protease L27V constructs were expressed in HEK293 cells. 1ng of DNA per well was transfected with 1. mu.g of vector DNA using the reverse transfection protocol described previously. Each cell sample was cultured in 1mL of medium in a 12-well plate. Cell lysates were prepared by removing the medium and adding 1mL of 1X PLB. Samples were diluted 1:10 in 1X PBS + 0.1% gelatin. A40. mu.L dilution sample was set up for TEV digestion. mu.L of the digestion reaction was added to 40. mu.L of DMEM without phenol red and 50. mu.L of PBI-3939 as previously described. FIG. 73H shows the luminescence of different CP-TEV protease L27V constructs expressed in HEK293 cells.

The data in fig. 73A-H indicate that the L27V variant can be cyclically swapped at different sites, and that different sites have different dependencies relating to tether length. Mammalian cell data and wheat germ data show similar fold reductions using TEV lysis. The more dependent chain, i.e., CP L27V variant that is more susceptible to TEV protease cleavage, is a potential candidate for PCA. Less tether-dependent CP L27V variants are likely potential candidates for self-complementation/dimerization assays.

Example 49 protein complementation assay

Protein Complementation Assay (PCA) provides for the detection of the interaction of two biological molecules, i.e., polypeptidesA tool of action. PCA utilizes two fragments of the same protein, e.g., enzyme, which can be reconstituted into a functionally active protein when brought into close proximity to each other. The OgLuc variants of the invention can be divided into two fragments at the site of tolerance for segregation. Each fragment of the isolated OgLuc variant can then be fused to one of a pair of polypeptides of interest (e.g., FKBP and FRB) that are thought to interact. If the two polypeptides of interest do in fact interact, the OgLuc fragments are in close proximity to each other and reconstitute a functionally active OgLuc variant. The activity of the reconstituted OgLuc variant can then be detected and measured using either the native or known coelenterazine or the novel coelenterazine of the invention. In another example, spliced OgLuc variants can be used in a more general complementation system similar to lac-Z (Langley et al, PNAS, 72: 1254-. In particular, an OgLuc variant fragment known to be complementary to another OgLuc variant fragment ("B") (designated "a") can be fused to a target protein, and the resulting fusion monitored by luminescence in the cells or cell lysates that contain the fragment B. Fragment B may be derived from the same cell (either in the chromosome or on another plasmid), or it may be a lysate or purified protein from another cell. Using fragment B and a polypeptide such as can be attached to a solid support

Can capture or immobilize the same fusion protein (fragment A). Luminescence can then be used to demonstrate successful capture or to quantify the amount of captured material. The manufacturer-specific protocol for protein complementation can be performed according to U.S. published application No. 2005/0153310, which is incorporated herein by reference.

1. The 9B8 opt PCA construct was generated as follows:

-p9B8PCA 1/4 ═ pF5A/Met- [9B8 opt (51-169) ] -GGGGSGGGSS-FRB (SEQ ID NO:331 and 332) & pF5A/FKBP-GGGSSGGGSG- [9B8 opt (1-50) ] (SEQ ID NO:337 and 338)

-p9B8PCA 1/2 ═ pF5A/Met- [9B8 opt (51-169) ] -GGGGSGGGSS-FRB (SEQ ID NO:331 and 332) & pF5A/[9B8 opt (1-50) ] -GGGGSGGGSS-FRB (SEQ ID NO:333 and 334)

-p9B8PCA 2/3 ═ pF5A/[9B8 opt (1-50) ] -GGGGSGGGSS-FRB (SEQ ID NO:333 and 334) & pF5A/FKBP-GGGSSGGGSG- [9B8 opt (51-169) ] (SEQ ID NO:335 and 336)

-p9B8PCA 3/4 ═ pF5A/FKBP-GGGSSGGGSG- [9B8 opt (51-169) ] (SEQ ID NOS: 335 and 336) & pF5A/FKBP-GGGSSGGGSG- [9B8 opt (1-50) ] (SEQ ID NOS: 337 and 338)

According to manufacturer's instructions

HD PCA constructs were transfected into HEK293 cells (15,000 cells/well) in 96-well plates. Then 5% CO at 37 ℃₂Cells were incubated overnight. After transfection, the medium on the cells was treated with CO-independent medium containing 10% FBS ₂The medium of (3) is replaced. Assay reagents containing 20. mu.M PBI-3939 were then added and luminescence was measured on Varioskan Flash at 28 ℃. Then 100 μ M rapamycin was added to the wells and luminescence was measured continuously for 1 hour. Fold response was calculated by dividing all luminescence of a given well by the rapamycin pretreated luminescence of the same well (fig. 74).

2. To demonstrate the use of the OgLuc variant in PCA, the L27V02A variant fragment was complemented with FKBP or FRB, and the interaction between FKBP and FRB was measured.

Table 42 lists the different Protein Complementation (PCA) constructs generated and tested. "2/3" represents a variant complement pair, wherein 1) the "old" C-terminus of L27V02A (the "old" ═ original C-terminus of L27V 02A) is the C-terminal partner of FKBP; and 2) the "old" N-terminus of L27V02A is the N-terminal partner of FRB. "1/4" represents a variant pair in which 1) the "old" N-terminus of L27V02A is the C-terminal partner of FKBP; and 2) the "old" C-terminus of L27V02A is the N-terminal partner of FRB. For all constructs, FKBP was located at the N-terminus of the L27V02A fragment, and FRB was located at the C-terminus of the L27V02A fragment. For example, PCA constructs were generated having splice sites at positions 157 (see tables 42, "2/3" and "1/4" # s 11 and 12(SEQ ID NO: 288-. Additional PCA constructs (SEQ ID NOS: 343-426 and 428-440) were generated (see Table 21).

Watch 42

TABLE 21

The complementary pair described in table 42 was cloned into the pF4Ag vector as previously described. The PCA construct (900. mu.L) was then expressed in rabbit reticulocyte lysate (RRL; Promega Corp.) or wheat germ extract (Promega Corp.). mu.L of the expression reaction for each PCA pair was mixed with 10. mu.L of 2 Xbinding buffer (100mM HEPES, 200mM NaCl, 0.2% CHAPS, 2mM EDTA, 20% glycerol, 20mM DTT, pH 7.5) and 7.5. mu.L of water, and 18. mu.L was transferred to wells of a 96-well plate. 2 μ L of 5 μ M rapamycin (final concentration 0.5 μ M) was added and incubated at room temperature for 10 min.

After incubation, 100 μ L of PBI-3939(50X stock diluted to 1X in assay buffer) was added and incubated at room temperature for 3 minutes. Luminescence was measured as previously described (fig. 76A-B: wheat germ; fig. 76C-D: rabbit reticulocytes; fig. 76E-F: cell-free system [ which system.

FIGS. 76A-G show the luminescence of different pairs of Protein Complements (PCA) L27V: fusing one L27V fragment of each pair with FKBP or FRB using either the 2/3 configuration (fig. 76A and 76C) or the 1/4 configuration (fig. 76B and 76D) as described, and the interaction of FKBP and FRB monitored in wheat germ extract (fig. 76A and 76B) and Rabbit Reticulocyte Lysate (RRL) (fig. 76C and 76D); luminescence with different Protein Complementation (PCA) negative controls (fig. 76E). Luminescence of the different protein-complementary L27V configured with 1/4 was measured in cell-free systems (RRL) (fig. 76F) and HEK293 cells (fig. 76G). The data in fig. 76A-G indicate that a number of different deletions (i.e., small fragments of the L27V variant) are functional.

3. To demonstrate the utility of the PCA constructs for cell-based PCA, the constructs were transfected into HEK293 cells and assayed using PBI-4377. Plasmid DNA (5ng) from each PCA pair (6, 12, 55, 84 and 103) was ligated with 40ng vector DNA (pGEM-3fz) and 5. mu.L of OPTI-

The phases were mixed and incubated for 5 minutes at room temperature. Then adding

HD (0.15. mu.L) and incubated again at room temperature for 15 minutes. DNA transfection mixture was added to 100 μ L HEK293 cells (1.5X 10) in DMEM (antibiotic-free) with 10% FBS⁵Individual cells/mL) and transferred to wells of a 96-well plate at 37 ℃ with 5% CO₂Incubate overnight.

After transfection, the medium was removed and used with CO-independent media containing 20. mu.M or 50X PBI-4377₂In the absence of CO₂Incubate at 37 ℃ for 2 hours with conditioning. Luminescence was measured, 10. mu.L of rapamycin was added, and luminescence was measured again every 2 minutes for 2 hours (FIGS. 76A-C).

4. To demonstrate the utility of PCA constructs for identifying inhibitors of protein-protein interactions, the construct described in #2 of this example was used.

The complementary pairs 103 "2/3", 157 "2/3", 103 "1/4" and 157 "1/4" described in table 42 were cloned into the pF4Ag vector as previously described. Then in rabbits according to the manufacturer's instructions The PCA construct (25. mu.L) was expressed in reticulocyte lysates (RRL; Promega Corp.). mu.L of the expression reaction for each PCA was mixed with 10. mu.L of 2 Xbinding buffer (100mM HEPES, 200mM NaCl, 0.2% CHAPS, 2mM EDTA, 20% glycerol, 20mM DTT, pH 7.5) and 7.5. mu.L of water, and 16.2. mu.L was transferred to wells of a 96-well plate. Rapamycin was examined with varying amounts of FK 506. To this reaction was added FRB-FKBP binding inhibitor, FK506(10 ×), and the reaction was incubated at room temperature for 10 minutes. Rapamycin (10 × stock solution) at 15nM was added to obtain a final concentration of rapamycin of 1.5nM and incubated for 2 hours at room temperature. After incubation, 100 μ L of PBI-3939(50X stock solution diluted to 1X in assay buffer) was added and incubated at room temperature for 3 minutes. In that

Luminescence was measured on a luminometer. Fig. 77 demonstrates that PCA constructs disclosed herein can be used to identify inhibitors of protein-protein interactions.

5. To demonstrate the utility of the cleaved form of the PCA constructs, the complementary pairs 103 "2/3", 157 "2/3" and 103 "1/4" were transfected into HEK293 cells and assayed using PBI-3939. 0.5ng of plasmid from each PCA pair was combined with 5. mu.L of OPTI-

And 49ng pGEM-3zf (Promega Corp.). The sample mixture was incubated at room temperature for 5 minutes. Then 0.15. mu.L of the extract was added

HD was added to the sample mixture and incubated at room temperature for 15 minutes. 100 μ L HEK293 cells in DMEM with 10% FBS at 1.5X10⁵Individual cells/mL were added to each sample mixture. The cell samples were then transferred to wells of a 96-well plate and incubated at 37 ℃ in 5% CO₂Incubate overnight.

The following day, 11.1 μ L of 10 μ M rapamycin (final concentration 1 μ M) was added to one half of the wells and 11.1 μ L of water was added to the other half of the wells. The 96-well plates were incubated at 37 ℃ for 1 hour. Will measure 100. mu.LFixative reagent + PBI-3939 (2. mu.L of 50X PBI-3939 mixed with 98. mu.L assay reagent as previously described) was added to each well and the plates were incubated for 4 minutes at 37 ℃. At 37 ℃ in

Luminescence was measured on the luminometer with an integration time of 0.5s and a reading of 1. (FIG. 76H).

Example 50 OgLuc cAMP Bioreceptors

The OgLuc variants of the invention can be linked to the light output not only by concentration, but also by modulation of the enzyme activity. For example, cAMP bioreceptors are developed by incorporating cAMP-binding domains from protein kinase a into the circulating exchanged OgLuc variants. The OgLuc variants of the invention can be cyclically swapped at sites that are tolerant to crossover by methods known in the art (e.g., U.S. published application No. 2005/0153310). The resulting cyclically exchanged OgLuc variant chimeric protein functions as an intracellular biological receptor for cAMP when expressed in mammalian cells. After cAMP binds to the biological receptors, the biological receptors undergo a conformational change, producing active luciferase. Treatment of cells with adenylyl cyclase forskolin should result in an increase in luminescence with increasing concentrations of forskolin. Similar biological sensors for targets including, but not limited to, calcium (Ca +2), cGMP and proteases such as caspases and Tobacco Etch Virus (TEV) can be developed by incorporating the appropriate binding domains or cleavage sites of each into the circulating exchanged OgLuc variant.

The use of OgLuc as a biological receptor was demonstrated by analysis of the variant 9B8 opt in the context of cAMP receptors. Constructs containing circular crossovers of the RII β B subunit of protein kinase a flanked by OgLuc variant sequences were generated and expressed in a cell-free system as described in PCT application PCT/US2007/008176, except that sites for circular crossovers were selected as described below. Nascent protein was measured in the presence and absence of cAMP. The response to cAMP was determined as the ratio of active (+) cAMP/(-) cAMP.

Based on the similarities to certain fatty acid binding proteins of known structure, previously described in PCT/US2010/33449, a structural model for OgLuc has been constructed. The model predicts the ordered sequence of the standard protein structural motif; alpha-helices and beta-sheets. Regions that transition between these structural elements were selected as cyclic exchange sites (see table 43).

1. The template for expression of the bioreceptor construct consists of: the C-terminal OgLuc sequence-RII. beta.B sequence-N-terminal OgLuc sequence in plasmid pF5(Promega Corp.).

T7 Coupled Wheat Germ Extract System (Promega Part # L4140) was used to translate the construct.

The Wheat Germ Extract Reaction contained 25. mu.L

Wheat Germ Extract(L411A)，2μL

Reaction Buffer(L462A)，1μL Amino Acid Mixture，Complete(L446A)，1μL

(40U/μL)(N2615)，1μL

T7 RNA polymerase (L516A), 1.0. mu.g DNA template and increasing the total volume to 50. mu.L with nuclease-free water. The reaction mixture was incubated at 30 ℃ for 120 minutes.

By adding 50. mu.L of OgLuc Glo Reagent (100mM MES (pH 6.0), 1mM CDTA, 150 mM KCl, 35mM thiourea, 2mM DTT, 0.25% to 50. mu.L of OgLuc translation mixture with or without 100. mu.M cAMP

NP-9(v/v)， 0.025％

DF 204 and 20 μ M PBI-3939) for OgLuc activity assay and kinetic readings for 30 minutes (

F500 plate reader). The response was determined by dividing the luminescence generated by the biological sensors containing cAMP by the luminescence generated by the biological sensors without cAMP (table 43).

Table 43: response of cyclically exchanged OgLuc biological receptors to cAMP

CP site	Answering
			27	2.6X
51	2.2X
		84	1.5X
122	4.3X
		147	1.9X
157	5.6X

2. At CP site 51 as described in 1Circularly permuted cAMP biotceptors of 9B8opt were constructed. And then utilized according to the manufacturer's instructions

HD BioSensors were transfected into HEK293 cells (15,000 cells/well) in 96-well plates and 5% CO at 37 ℃₂Incubation was performed overnight. After transfection, the medium was removed and CO-independent with 10% FBS₂The medium of (3) is replaced. Then 5% CO at 37 ℃₂Cells were incubated for 2 hours, after which different concentrations of FSK were added. Then again at 37 ℃ with 5% CO ₂Cells were incubated for 3 hours. Then 6 μ M PBI-3939 was added and the luminescence was measured after 13 minutes (FIG. 78).

3. Cyclic exchanged ("CP"; e.g., CP6 refers to the old residue at position 6 followed by the new residue at position 1 after methionine) and directly spliced ("SS"; e.g., SS6 refers to the receptor located as L27V in the form of the OgLuc (1-6) -RII β b binding site (SEQ ID NOS: 441 and 442) -OgLuc (7-169)) were used as cAMP biological receptors (SEQ ID NO: 467-. The CP (SEQ ID NO:467-498 and 555-574) and SS (SEQ ID NO:499-554) versions of the L27V variant were derived as previously described and expressed in rabbit reticulocyte lysate (RRL; Promega Corp.) according to the manufacturer's instructions. The linker sequence between the C-terminus of the RII β b binding site and the OgLuc luciferase sequence is GGGTCAGGTGGATCTGGAGGTAGCTCTTCT (SEQ ID NO: 575). Linker sequence between the N-terminus of the RII β b binding site and the OgLuc luciferase sequence AGCTCAAGCGGAGGTTCAGGCGGTTCCGGA (SEQ ID NO:576) 3.75 μ L of the expression reaction was mixed with 1.25 μ L of 4X cAMP (final concentration 1nM to 0.1mM) and incubated for 15 min at room temperature. After incubation, 100 μ L of PBI-3939(50X stock solution diluted 1X in assay buffer) was added and incubated for 3 minutes at room temperature. In that

Luminescence was measured on a luminometer (FIGS. 79A-B). The luminescence of CP and SS forms of the L27V variant expressed in HEK293 cells and treated with forskolin as previously described was also measured (fig. 79C-D). FIGS. 79A-D show recurrent crossing of OgLuc variants disclosed hereinAlternative and directly spliced forms are useful as biological sensors.

Example 51 subcellular distribution and localization

To analyze subcellular distribution, U2OS cells were plated at 2x10⁴Individual cell/cm²The cells were inoculated into 10% FBS-containing McCoy's 5A medium (Invitrogen) in a glass-bottom petri dish. Then 5% CO at 37 ℃₂Cells were incubated for 24 hours. Then 1/20 volumes of transfection mixture were used (

HD and pF5A-CMV-L27V (L27V variant (SEQ ID NO:88)) or pGEM3ZF (Promega Corp.; negative control)) cloned into pF5A vector containing CMV promoter (Promega Corp.) were transfected into cells and 5% CO at 37 deg.C₂Incubate for 24 hours. After incubation, CO-independent cultures containing 0.5% FBS and 100. mu.M PBI-4378 were used₂Replacing the cell culture medium. After 30 min incubation at 37 ℃, unfiltered images were captured on an Olympus LV200 bioluminescent microscope using a 60X eyepiece (fig. 80A-B) for 25, 100, 1000 and 5000 ms.

To analyze subcellular localization, an N-terminal L27V fusion containing GPCR AT1R (angiotensin type 1 receptor (SEQ ID NOs: 459 and 460)) with IL-6 secretory sequences (SEQ ID NOs: 461 and 462) or transcription factor Nrf2(SEQ ID NO:317) was generated using GSSG linkers (SEQ ID NOs: 457 and 458) and transfected into U2OS cells as previously described (fig. 81A-C). Figure 81C ("GPRC") shows expression of the construct with IL6 signal sequence upstream of the L27V variant sequence and AT1R downstream of the L27V variant sequence. The L27V variant alone ("unfused") was also transfected. At 37 deg.C, 5% CO ₂After 24 hours of incubation, the cells were incubated with 0.5% FBS-independent CO₂The medium of (a) replaces the cell culture medium and is at 37 ℃ in the absence of CO₂The atmosphere was adjusted to equilibrate for 1 hour. Equal volume of medium +200 μ M PBI-3939 was then added and unfiltered images were immediately captured on an Olympus LV200 bioluminescence microscope using either a 60X or 150X eyepiece (fig. 81A-C). For cells expressing L27V alone, PBI-3939 was washed off the cells and images were immediately captured.

Example 52 monitoring intracellular signaling pathways

This example provides two examples of novel luciferases useful for monitoring intracellular signalling pathways at the protein level (in contrast to the example of response elements representing transcriptional activation). Variant 9B8opt (SEQ ID NO:24) was fused (at the C-terminus, i.e., N-IkB- (9B8opt) -C)) to IkB (Gross et al, Nature Methods 2(8):607-614(2005)) or to ODD (the oxygen-dependent degradation domain of Hif-1-. alpha. (Moroz et al, PLoS One 4(4): e5077(2009)) at the N-terminus, i.e., N- (9B8opt) -ODD-C)). IKB is known to degrade in cells following stimulation with TNF α; thus, the IKB- (9B8opt) construct can be used as a receptor for live cell TNF α. ODD (Hif-1-. alpha.) is known to accumulate in cells after stimulation with hypoxia-inducing compounds; ODD- (9B8opt) can therefore be used as a hypoxia receptor for living cells.

Constructs containing fusions of IkB or ODD with 9B8opt (pF5A) were expressed in HEK293 cells by reverse transfection (5ng (IkB) or 0.05 ng (ODD) DNA mixed with vector DNA to give a total of 50ng) as previously described and 5% CO at 37 ℃. (5% CO)₂Incubate for 24 hours. After transfection, fresh CO-independent medium containing 0.5% FBS and 20. mu.M PBI-4377 was used₂The medium of (3) is substituted for the medium and is subjected to CO at 37 deg.C₂The atmosphere was equilibrated for 4 hours. The cells are then exposed to stimuli: TNF α was used for IkB fusion expressing cells and phenanthroline was used for ODD fusion expressing cells. DMSO (vehicle) was added to control cells. For the TNF α/IKB samples, 100 μ g/mL cycloheximide was added approximately 15 minutes before the addition of the stimulus to prevent synthesis of new proteins. After treatment at the indicated time points, the luminescence of the cells was measured. For data normalization, RLU for each sample at a given time point was divided by RLU from the same sample immediately following stimulation. The fold response of each receptor was then determined (fig. 82A-C).

B. L27V was used to monitor oxidative stress signaling pathways at the protein level. L27V or Renilla luciferase (Rluc) was fused to Nrf2/NFE2L2 in the pF5K expression vector (at the C-terminus; i.e., N-Nrf2- (L27V) -C or N-Nrf2- (Rluc) -C). Keap1 is a negative regulator of Nrf2(SEQ ID NO: 217). To faithfully represent the regulation of Nrf2-L27V02 protein levels, Keap1 was co-expressed to maintain Nrf2 low levels (via ubiquitination).

Expression of Nrf2-L27V or Nrf2-Rluc (5ng, pF5K) and in HEK293 cells by simultaneous transfection of cells when seeded into 96-well plates as previously described

-Keap1 fusion protein (pFN21-HT7-Keap1(SEQ ID NO: 316); 50ng) and 5% CO at 37 ℃%₂Incubate for 24 hours. After transfection, the cells were transfected with 0.5% FBS and 20. mu.M PBI-4377 (for L27V) or 20. mu.M ENDUREN^TMFresh CO-independent (Promega Corp.) (for Renilla luciferase)₂The medium of (3) replaces the medium and is in CO at 37 ℃₂Cells were equilibrated in atmosphere for 4 hours. For kinetic analysis, 20 μ M D, L sulforaphane or vehicle (DMSO) was used. In fig. 83A, the post-treatment measurement luminescence is measured at the specified time point as previously described. For data normalization, the luminescence of each sample at a given time point was divided by the luminescence from the same sample immediately after stimulation (fig. 83B-C).

C. Comparisons of responses of Nrf2 receptors described in B and Nrf2(ARE) -Luc2P reporter (Promega Corp.) were performed. Nrf2 sensors and reporters were screened as previously described in section B above. For firefly (Luc2P) reporter gene assay, ONE-GLO was used^TMAnd (4) measuring the reagent. FIGS. 84A-B provide normalized response at 2 hours for Nrf2-L27V and normalized response at 16 hours for Nrf2(ARE) -Luc 2P.

Example 53 evaluation of OgLuc variants as bioluminescent reporter by BRET

Bioluminescence Resonance Energy Transfer (BRET) allows monitoring of protein-protein interactions. Intramolecular energy transfer was examined between the IV and HT7 fusion partners, in which HT7 had previously been labeled with a fluorophore, either TMR (excitation/emission wavelength 555/585nm) or rhodamine 110 (excitation/emission wavelength 502/527 nm). mu.L of bacterial cell lysate containing the IV-HT7 fusion protein of example 34 was incubated with 0.001-10. mu.M fluorophore ligand or no ligand for 1 hour at room temperature. After incubation, the cells containing 22. mu.M coelenterazine-h 50. mu.L of RENILLA-GLO^TMAdd to 50 μ L of enzyme-ligand mixture and record the emission wavelength at 5 min. The wavelengths of the examples of IV-HT7 with TMR (fig. 83A) or rhodamine 110 ("Rhod 110") (fig. 85B) are shown, indicating that BRET is larger, i.e., larger with rhodamine 110, when the excitation/emission of the ligand is close to the 460nm luminescence peak of OgLuc. This data shows that intramolecular energy transfer on the fusion protein can occur between the OgLuc variant and the fluorophore. Three different controls were used for comparison (data not shown): 1) no HT fusion, 2) HT-fusion not labeled with HT ligands, and 3) labeled HT-fusion that is proteolytically cleaved at the TEV site between OgLuc and HT (which indicates the proximity/distance involved). No BRET was observed in the three different controls, indicating that HT was involved in achieving BRET. BRET is greater for C1+ A4E and IV with C-terminal HT7 compared to N-terminal HT 7.

Example 54 protein proximity assay in live cell or lysed form

In one example, Circularly Permuted (CP) or directly spliced (SS) OgLuc fusion proteins were applied for measurement of protein proximity. The OgLuc is exchanged or spliced by insertion of a protease substrate amino acid sequence (e.g., TEV) to generate low bioluminescence. Inactive luciferase is tethered (e.g., via gene fusion) to the monitor protein. The potential interacting proteins are tethered (e.g., via genetic fusion) to a protease (e.g., TEV). When the two monitoring proteins interact or are in sufficient proximity (e.g., via a constitutive interaction, drug stimulus or pathway response), the luciferase is cleaved to produce increased bioluminescent activity. This example can be applied to the measurement of protein proximity in cells or in biochemical assays. Also, the high thermal stability of the OgLuc variant luciferase may enable measurement of antibody-antigen interactions in lysed cells or biochemical assays.

Example 55 bioluminescence assay

1. To determine the use of the OgLuc variant in a bioluminescent assay for detecting caspase-3, the 9B8 opt variant was used for growth using a propelloenteron substrate comprising a DEVD caspase-3 cleavage sequence And (4) performing bioluminescence measurement. Purified caspase-3 was mixed with a sample of E.coli lysate expressing variant 9B8 opt, prepared as described in example 27, diluted 10 fold in a solution containing 100mM MES pH 6.0, 1 mM CDTA, 150mM KCl, 35mM thiourea, 2mM DTT, 0.25%

NP-9(v/v)，0.025％

DF 204, with or without 23.5. mu. M z-DEVD-coelenterazine-h in 100mM HEPES pH 7.5 buffer. Caspase-3 was incubated with lysate samples for 3 hours at room temperature and at different time points in Turner MODULUS^TMLuminescence was detected on a luminometer. Samples containing only bacterial lysate and only caspase-3 were used as controls. Three replicates were used. Fig. 86 and table 44 demonstrate that 9B8 opt, and thus other OgLuc variants of the invention, can utilize a pre-coelenterazine substrate in a bioluminescence assay to detect an enzyme of interest.

Table 44: mean luminescence in RLU generated from bacterial lysates expressing 9B8 opt incubated with or without purified caspase-3 using z-DEVD-coelenterazine-h as substrate.

Time (minutes)	Caspase-free (RLU)	+ caspase (RLU)
			5	26,023	25,411
15.3	7,707	36,906
			29.9	4,013	41,854
60.9	2,305	43,370
			190.3	1,155	42,448

2. The L27V variant was used in a bioluminescent assay using a pro-luminal enteron substrate comprising a DEVD caspase-3 cleavage sequence. Purified caspase-3 (1mg/mL) in 100mM MES pH 6 (50. mu.L) was mixed with 227nM L27V02 variant and 47. mu.M PBI-3741 (z-DEVD-coelenterazine-h) in assay buffer (50. mu.L). The reaction was incubated at room temperature for 3 hours and the luminescence was detected as previously described. Comparing the assay using the L27V variant with the assay in the form of firefly luciferase, CASPASE-

3/7-assay System (Caspase-Glo; Promega Corp.). Table 45 shows that the L27V variant, and thus other OgLuc variants of the invention, can utilize the pre-coelenterazine substrate in a bioluminescent assay to detect the enzyme of interest.

TABLE 45

Example 56 immunoassay

The OgLuc variants of the invention can be incorporated into a variety of different immunoassay concepts. For example, the OgLuc variants are genetically fused or chemically conjugated to a primary or secondary antibody to provide a detection method for a particular analyte. As another example, the OgLuc variant is fused or chemically conjugated to protein a, protein G, protein L, or any other peptide or protein gene known to bind Ig fragments, and this can then be used to detect a particular antibody that binds to a particular analyte. As another example, the OgLuc variants are fused or chemically conjugated to the streptavidin gene and used to detect specific biotinylated antibodies that bind to specific analytes. As another example, complementary fragments of the OgLuc variant are genetically fused or chemically conjugated to a primary antibody and a secondary antibody, wherein the primary antibody recognizes a specific immobilized analyte and the secondary antibody recognizes the primary antibody, both in an ELISA-like format. The OgLuc variant activity, i.e., luminescence, reconstitutes in the presence of the immobilized analyte and serves as a tool for quantifying the analyte.

As another example, a complementary pair of OgLuc variants can be fused to two antibodies, wherein one antibody recognizes a particular analyte at one epitope and the second antibody recognizes an analyte at a separate epitope. The OgLuc variant activity will reconstitute in the presence of the analyte. The method will be amenable to the measurement of analyte quantification in complex environments such as cell lysates or cell culture media. As another example, a complementary fragment of the OgLuc variant will be fused to two antibodies, wherein one antibody recognizes a particular analyte, whether modified or not, and the secondary antibody recognizes only the modified analyte (e.g., post-translational modification). The OgLuc variant activity will reconstitute only when its modified analyte is present. The method will be amenable to complex environments such as measurement of modified analytes in cell lysates. As another example, the OgLuc variant will be used in a competitive sandwich ELISA format in combination with an analyte (e.g., prostaglandin) conjugate.

Example 57 dimerization assay

This example demonstrates that full-length circularly permuted OgLuc variants can be fused to corresponding binding partners, e.g., FRB and FKBP, and used in protein complementation type assays. A key difference between the manufacturer-based protocol disclosed herein and traditional protein complementation is that there is no complementation, but rather dimerization of two full-length enzymes (e.g., circularly-swapped OgLuc variants).

Briefly, a protein reporter configured for low activity, cyclic exchange was simply fused to both of the fusion protein partners (see fig. 87A). For example, each fusion partner may be linked to an identical structure, exchanged reporter. The interaction of the fusion partners brings the exchanged reporters in close proximity, allowing reconstitution of the hybridized reporters with greater activity. The novel hybridization reporter includes portions of the reporter exchanged per cycle in a manner that reduces structural limitations.

Cloning of the circularly permuted, directly spliced L27V variants CP84 and CP103(N- (SS-169) - (1-SS) as described previously according to the manufacturer's instructions₁) -FRB-C and C- (1-SS)₁) - (SS-169) -FKBP) and in rabbit reticulocyte lysate (RRL; promega Corp.) (25 μ L). 1.25. mu.L of the expression reaction for each dimerization pair was mixed with 10. mu.L of 2 Xbinding buffer (100mM HEPES, 200mM NaCl, 0.2% CHAPS, 2 mM EDTA, 20% glycerol, 20mM DTT, pH 7.5) and 7.5. mu.L of water, and 18. mu.L was transferred to wells of a 96-well plate. To the reaction 2. mu.L of rapamycin (final concentration 0 and 0.1-1000nM) was added and the reaction was incubated at room temperature for 10 min. After incubation, 100 μ L PBI-3939(50X stock diluted to 1X in assay buffer) was added and incubated for 3 minutes at room temperature. In that

Luminescence was measured on a luminometer (fig. 87B) and the response determined (fig. 87C). Fig. 87B-C demonstrate that OgLuc variants of the invention can be used to detect protein-protein interactions by PCA-type dimerization assays.

Example 58 intracellular half-Life

The intracellular half-lives of the OgLuc variants 9B8, 9B8+ K33N, V2, L27V and V2+ L27M were determined. According to manufacturer's instructions

LT1(Mirus) CHO cells (500,000) in F12 medium containing 10% FBS and 1X sodium pyruvate in 15-100mm plates were transfected with 30. mu.L 100 ng/. mu.L plasmid DNA containing 9B8, 9B8+ K33N, V2, L27V ("V2 + L27V") or V2+ L27M (all in the pF4A vector background). The cells were then incubated for 6 hours.

After incubation, the medium was removed and 1mL of pancreatin was added to detach the cells from the plate. Then 3mL of F12 medium was added and the cells were counted. Cells were then seeded at 10,000 cells/well into 6 wells of a 96-well plate (6 wells/variant) and incubated overnight at 37 ℃. The samples were distributed to cover 3 plates. Each plate had 6 replicates of different time point measurements.

After overnight incubation, the medium was removed from the cells for the t-0 sample and 100 μ L of assay buffer (previously described; no substrate) was added. The samples were frozen on dry ice and stored at-20 ℃. In OPTI-

In (1) the cycloheximide (100mg/mL) was diluted 1:100 to a final concentration of 1 mg/mL. Also in OPTI-

In (1%) DMSO (100%) was diluted 1:100 (final concentration 1%). Diluted cycloheximide (1mg/mL) (11. mu.L) was added to 3 replicates of each transfected variant sample and 11. mu.L of diluted DMSO (1%) was added to the other 3 replicates. Then 5% CO at 37 ℃₂Cells were incubated and removed at various time points (i.e., 0, 0.5, 0.9, 2.5, 4.3, and 6.2 hours) and processed as t-0 samples.

For analysis, cells were thawed to room temperature and 10 μ L was assayed in 50 μ L assay reagent. In that

Luminescence was measured on a luminometer. At each time point, luminescence was measured for untreated and cycloheximide treated samples. RLU benefits by untreated cellsRLU of cells treated with cycloheximide were normalized.

The intracellular half-life of each variant was calculated at each time point by measuring the ratio of luminescence from Cycloheximide (CHX) treatment to untreated luminescence. The natural logarithm of the rate of the shift over time was then plotted (% treated vs. untreated) and the half-life calculated (table 46). The OgLuc variant has an intracellular half-life of about 6-9 hours with the full strength CMV promoter, but the half-life is reduced with the CMV deletion variant (d 2). The presence of PEST degradation signal in combination with the full strength CMV promoter significantly reduced the half-life.

TABLE 46

Sample (I)	CMV No degradation Signal	CMV d2 No degradation Signal	CMV Pest
				9B8	6.32	3.87	1.43
K33N	9.24	3.70	1.18
				V2	9.63	4.28	1.61
V2+L27V	6.66	4.78	1.63
				V2+L27M	8.89	6.98	1.63

Another experiment was performed using HEK293 cells using the reverse transfection protocol described in example 52 (data not shown). The results from this experiment indicate that the intracellular half-life of the L27V variant with PEST is 10 minutes. The L27V variant used in this experiment that did not contain a degradation signal showed no decay during the course of this experiment. In this case the attenuation was normalized to untreated cells when t is 0.

Example 59 Exposure of OgLuc variant to Urea

As firefly luciferase is known to be relatively unstable, it is more sensitive to urea exposure. To determine whether this is also the case for the OgLuc variant, the sensitivity of OgLuc to urea was determined. Mu.l of 45.3. mu. M L27V enzyme was mixed with 100. mu.L of urea solution (100 mM MOPS, pH 7.2, 100mM NaCl, 1mM CDTA, 5% glycerol and different concentrations of urea) and incubated for 30 min at room temperature. mu.L of urea + L27V enzyme solution was diluted 10,000 times to 0.1% phenol red free

50 μ L was mixed with 50 μ L of assay reagent (previously described) containing 100 μ M PBI-3939 and luminescence read at 10 minutes (FIG. 88). Fig. 88 shows that L27V resists urea or refolds very quickly into a functional enzyme after removal of urea. This indicates when chemical denaturing conditions are involved, e.g., in the OgLuc variant-based reaction L27V can be used as a reporter enzyme, using denaturation to stop multiple complexation in conditions for the enzymatic reaction.

0.31mg/mL of purified L27V variant stock solution was diluted 100,000 fold into buffer (PBS +1mM DTT + 0.005% IGEPAL) and incubated with 3M urea for 30 minutes at 25 ℃ and then mixed with assay reagent 1:1 (50. mu.L + 50. mu.L) containing 100. mu.M PBI-3939 (described previously). As described previously in

The reaction was measured on an F500 photometer (duration 100 min; 1 min reading interval) (fig. 89). The results show that 3M urea reduced the activity of the L27V variant by about 50%, but after 2-fold dilution of urea (to a final concentration of 1.5M), the activity increased, probably due to refolding.

Example 60 imaging of OgLuc fusion proteins

This example demonstrates the use of OgLuc and the OgLuc variants to monitor protein translocation in living cells without the need for fluorescence excitation. The OgLuc variants were fused to human glucocorticoid receptor (GR; SEQ ID NOS: 451 and 452), human protein kinase C α (PKCa; SEQ ID NOS: 449 and 450) or LC3(SEQ ID NOS: 577 and 578). To analyze subcellular protein translocation using bioluminescence imaging, HeLa cells were plated at 2x10⁴Individual cell/cm²The cells were inoculated into 10% FBS-containing DMEM medium in a glass-bottom culture dish (MatTek). Then incubated at 37 ℃ for 24 hours with 5% CO 2. Then using 1/20 volume transfection: (

HD and DNA encoding L27V02-GR (SEQ ID NOS: 453 and 454) or L27V02-PKC α (SEQ ID NOS: 455 and 456) mixtures cloned into pF5A vector (Promega Corp.). Plasmid DNA 1:20 for L27V02-GR was diluted into pGEM-3ZF (Promega Corp.) to obtain the appropriate expression level of L27V 02-GR. Undiluted DNA was used for L27V02-LC3 and L27V02-PKC α. Then 5% CO at 37 ℃₂Incubation underCells were cultured for 24 hours. Cells transfected with GR fusion protein were starved for GR agonists for 20 hours using MEM medium (Invitrogen) supplemented with 1% charcoal/dextran treated FBS. 24 hours post-transfection (for PKC. alpha. measurement) or 48 hours post-transfection (for GR measurement), CO-independent samples containing 100. mu.M PBI-3939 were used immediately before imaging₂The medium of (a) replaces the cell culture medium. Unfiltered images were immediately captured on an Olympus LV200 bioluminescent microscope using a 150X eyepiece.

Cytosolic nuclear translocation of the L27V02-GR fusion protein was achieved by stimulation with 0.5mM dexamethasone for 15 min. Cytosolic translocation of the L27V02-PKC α fusion protein to the plasma membrane was achieved by stimulation with 100nM PMA for 20 min. Cells transfected with L27V02-LC3 fusion protein were either untreated or treated with 50mM chloroquine in DMEM medium (Invitrogen) with 10% FBS.

L27V 02-glucocorticoid receptor

In the absence of glucocorticoid, the Glucocorticoid Receptor (GR) is complexed with Hsp90 protein and is localized in the cytosol. After GR interacts with glucocorticoids such as dexamethasone, GR proteins are detached from these protein complexes and translocated to the nucleus to regulate gene expression. FIGS. 90A-B show bioluminescence imaging of cytosolic to Nuclear Receptor (NR) translocation of L27V 02-Glucocorticoid Receptor (GR) fusion protein induced by the PBI-3939 substrate dexamethasone in HeLa cells.

L27V02-PKCa

After treatment with phorbol esters, PKC α proteins recruit to the plasma membrane and modulate cellular responses, including membrane dynamics and signal transduction. FIGS. 91A-B show bioluminescent imaging of phorbol ester-induced cytosolic translocation of protein kinase C α (PCK α) to the plasma membrane in U-2 OS cells using PBI3939 substrate OgLuc L27V02-PKC α fusion.

L27V-LC3

Dissociation of the processed LC-3 protein from the autophagosome represents a marker step in autophagy. Chloroquine treatment arrests the autophagy changes at this stage, resulting in accumulation of LC-3 protein on the autophagosomes (resulting in a punctate subcellular distribution). FIGS. 92A-B show bioluminescent imaging of chloroquine-induced autophagosome protein translocation using the PBI-3939 substrate OgLuc L27V-LC3 fusion protein (SEQ ID NOS: 592 and 593) in two representative HeLa cell samples.

Watch 47 attached watch

Sequence listing

SEQ ID NO:1 (Natural mature OgLuc protein) Cereus elegans

FTLADFVGDWQQTAGYNQDQVLEQGGLSSLFQALGVSVTPIQKVVLSGENGLKADIHVIIPYEGLSGFQ MGLIEMIFKVVYPVDDHHFKIILHYGTLVIDGVTPNMIDYFGRPYPGIAVFDGKQITVTGTLWNGNKIY DERLINPDGSLLFRVTINGVTGWRLCENILA

SEQ ID NO 2(C1A4E nucleotide)

ATGGTGTTTACATTGGAGGATTTCGTTGGAGACTGGCGGCAGACAGCTGGATACAACCAAGATCAAGTG TTAGAACAAGGAGGATTGTCTAGTCTGTTCCAAAAGCTGGGAGTGTCAGTCACCCCAATCCAGAAAATT GTGCTGTCTGGGGAGAATGGGTTAAAATTTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGT TTTCAAATGGGTCTGATTGAAATGATCTTCAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGATT ATTCTCCATTATGGTACACTCGTTATTGACGGTGTGACACCAAACATGATTGACTACTTTGGACGCCCT TACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAGTTACTGGAACTCTGTGGAACGGCAACAAG ATCATTGATGAGCGCCTGATCAACCCAGATGGTTCACTCCTCTTCCGCGTTACTATCAATGGAGTCACC GGATGGCGCCTTTGCGAGCGTATTCTTGCC

SEQ ID NO 3(C1A4E protein)

MVFTLEDFVGDWRQTAGYNQDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKFDIHVIIPYEGLSG FQMGLIEMIFKVVYPVDDHHFKIILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILA

SEQ ID NO. 4(QC27 nucleotides)

ATGGTGTTTACATTGGAGGATTTCGTTGGAGACTGGCGGCAGACAGCTGGATACAACCTAGATCAAGTG TTAGAACAAGGAGGATTGTCTAGTCTGTTCCAAAAGCTGGGAGTGTCAGTCACCCCAATCCAGAAAATT GTGCTGTCTGGGGAGAATGGGTTAAAAATTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGT TTTCAAATGGGTCTGATTGAAATGATCTTCAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGATT ATTCACCATTATGGTACACTCGTTATTGACGGTGTGACACCAAACATGATTGACTTCTTTGGACGCCCT TACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAGTTACTGGAACTCTGTGGAACGGCAACAAG ATCATTGATGAGCGCCTGATCAACCCAGATGGTTCACTCCTCTTCCGCGTTACTATCAATGGAGTCACC GGATGGCGCCTTTGCGAGCGTATTCTTGCC

SEQ ID NO 5(QC27 protein)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSG FQMGLIEMIFKVVYPVDDHHFKIIHHYGTLVIDGVTPNMIDFFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILA

SEQ ID NO 6(QC 279 a nucleotides)

ATGGTGTTTACATTGGAGGATTTCGTTGGAGACTGGCGGCAGACAGCTGGATACAACCTAGATCAACTG TTAGAACAAGGAGGATTGTCTAGTCTGTTCCAAAAGCTGGGAGTGTCAGTCACCCCAATCCAGAAAATT GTGCTGTCTGGGGAGAATGGGTTAAAAATTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGT TATCAAATGGGTCAGATTGAAAAGATCTTCAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGATT ATTCGCCATTATGGTACACTCGTTATTGACGGTGTGACACCAAACATGATTGACTTCTTTGGACGCCCT TACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAGTTACTGGAACTCTGTGGAACGGCAACAAG ATCATTGATGAGCGCCTGATCAACCCAGATGGTTCACTCCTCTTCCGCGTTACTATCAATGGAATCACC GGATGGCGCCTTTGCGAGCGTATTCTTGCC

SEQ ID NO 7(QC 279 a protein)

MVFTLEDFVGDWRQTAGYNLDQLLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSG YQMGQIEKIFKVVYPVDDHHFKIIRHYGTLVIDGVTPNMIDFFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGITGWRLCERILA

SEQ ID NO 8(IVY nucleotide)

ATGGTGTTTACATTGGAGGATTTCGTTGGAGACTGGCGGCAGACAGCTGGATACAACCAAGATCAAGTG TTAGAACAAGGAGGATTGTCTAGTCTGTTCCAAAAGCTGGGAGTGTCAGTCACCCCAATCCAGAAAATT GTGCTGTCTGGGGAGAATGGGTTAAAAATTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGT TTTCAAATGGGTCTGATTGAAATGATCTACAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGGTT ATTCTCCATTATGGTACACTCGTTATTGACGGTGTGACACCAAACATGATTGACTACTTTGGACGCCCT TACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAGTTACTGGAACTCTGTGGAACGGCAACAAG ATCATTGATGAGCGCCTGATCAACCCAGATGGTTCACTCCTCTTCCGCGTTACTATCAATGGAGTCACC GGATGGCGCCTTTGCGAGCGTATTCTTGCC

SEQ ID NO 9(IVY protein)

MVFTLEDFVGDWRQTAGYNQDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSG FQMGLIEMIYKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILA

SEQ ID NO 10(IVY + C1.3 nucleotides)

ATGGTGTTTACATTGGAGGATTACGTTGGAGACTGGCGGCAGACAGCTGGATACAACCAAGATCAAGTG TTAGAACAAGGAGGATTGTCTAGTCTGTTCCAAAATCTGGGAGTGTCTGTCACCCCAATCCAGAAAATT GTGCTGTCTGGGGAGAATGGGTTAAAAATTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGT TTTCAAATGGGTCTGATTGAAATGATCTACAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGGTT ATTCTCCATTATGGTACACTCGTTATTGACGGTGTGACACCAAACATGATTGACTACTTTGGACGCCCT TACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAGTTACTGGAACTCTGTGGAACGGCGACAAG ATCATTGATGAGCGCCTGATCAACCCAGATGGTTCACTCCTCTTCCGCGTTACTACCAATGGAGTCACC GGATGGCGCCTTTGCGAGCGTATTCTTGCC

11(IVY + C1.3 protein)

MVFTLEDYVGDWRQTAGYNQDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSG FQMGLIEMIYKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGDK IIDERLINPDGSLLFRVTTNGVTGWRLCERILA

SEQ ID NO 12(IVY C5.19 nucleotide)

ATGGTGTTTACATTGGAGGATTTCGTTGGAGACTGGCGGCAGACAGCTGGATACAACCAAGATCAAGTG TTAGAACAAGGAGGAGTGTCTAGTCTGTTCCAAAAGCTGGGAGTGTCAATCACCCCAATCCAGAAAATT GTGCTGTCTGGGGAGAATGGGTTAAAAATTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGT TTTCAAATGGGTCTGATTGAAATGATCTACAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGGTT ATTCTCCATTATGGTACACTCGTTATTGACGGTGTGACACCAAACATGATTGACTACTTTGGACGCCCT TACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAGTTACTGGAACTCTGTGGAACGGCAACAAG ATCATTGATGAGCGCCTGATCAACCCAGATGGTTCAATCCTCTTCCGCGTTACTATCAATGGAGTCACC GGATGGCGCCTTTGCGAGCGTATTCTTGCC

13(IVY C5.19 protein)

MVFTLEDFVGDWRQTAGYNQDQVLEQGGVSSLFQKLGVSITPIQKIVLSGENGLKIDIHVIIPYEGLSG FQMGLIEMIYKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSILFRVTINGVTGWRLCERILA

SEQ ID NO 14(IV nucleotides)

ATGGTGTTTACATTGGAGGATTTCGTTGGAGACTGGCGGCAGACAGCTGGATACAACCAAGATCAAGTG TTAGAACAAGGAGGATTGTCTAGTCTGTTCCAAAAGCTGGGAGTGTCAGTCACCCCAATCCAGAAAATT GTGCTGTCTGGGGAGAATGGGTTAAAAATTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGT TTTCAAATGGGTCTGATTGAAATGATCTTCAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGGTT ATTCTCCATTATGGTACACTCGTTATTGACGGTGTGACACCAAACATGATTGACTACTTTGGACGCCCT TACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAGTTACTGGAACTCTGTGGAACGGCAACAAG ATCATTGATGAGCGCCTGATCAACCCAGATGGTTCACTCCTCTTCCGCGTTACTATCAATGGAGTCACC GGATGGCGCCTTTGCGAGCGTATTCTTGCC

SEQ ID NO 15(IV protein)

MVFTLEDFVGDWRQTAGYNQDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSG FQMGLIEMIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILA

16 (nucleotide 15C 1) SEQ ID NO

ATGGTGTTTACATTGAAGGATTTCGTTGGAGACTGGCGGCAGACAGCTGGATACAACCAAGATCAAGTG TTAGAACAAGGAGGATTGTCTAGTCTGTTCCAAAATCTGGGAGTGTCAGTCACCCCAATCCAGAAAATT GTGCTGTCTGGGGAGAATGGGTTAAAAATTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGT TATCAAATGGGTCAGATTGAAAAGATCTTCAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGGTT ATTCTCCATTATGGTACACTCGTTATTGACGGTGTGACACCAAACATGATTGACTACTTTGGACGCCCT TACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAGTTACTGGAACTCTGTGGAACGGCAACAAG ATCATTGATGAGCGCCTGATCAACCCAGATGGTTCACTCCTCTTCCGCGTTACTATCAATGGAGTCACC GGATGGCGCCTTTGCGAGCGTATTCTTGCC

17(15C1 protein)

MVFTLKDFVGDWRQTAGYNQDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSG YQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILA

18 (nucleotide 9B 8)

ATGGTGTTTACATTGGAGGATTTCGTTGGAGACTGGCGGCAGACAGCTGGATACAACCTAGATCAAGTG TTAGAACAAGGAGGATTGTCTAGTCTGTTCCAAAAGCTGGGAGTGTCAGTCACCCCAATCCAGAAAATT GTGCTGTCTGGGGAGAATGGGTTAAAAATTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGT TATCAAATGGGTCAGATTGAAAAGATCTTCAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGGTT ATTCTCCATTATGGTACACTCGTTATTGACGGTGTGACACCAAACATGATTGACTACTTTGGACGCCCT TACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAGTTACTGGAACTCTGTGGAACGGCAACAAG ATCATTGATGAGCGCCTGATCAACCCAGATGGTTCACTCCTCTTCCGCGTTACTATCAATGGAGTCACC GGATGGCGCCTTTGCGAGCGTATTCTTGCC

SEQ ID NO 19(9B8 protein)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSG YQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILA

20 (nucleotide 9F 6)

ATGGTGTTTACATTGGAGGATTTCGTTGGAGACTGGCGGCAGACAGCTGGATACAACCTAGATCAAGTG TTAGAACAAGGAGGAGTGTCTAGTCTGTTCCAAAAGCTGGGAGTGTCAATCACCCCAATCCAGAAAATT GTGCTGTCTGGGGAGAATGGGTTAAAAATTGATATTCATGTCATCATCCCTTACGAGGGACTCAGTGGT TATCAAATGGGTCAGATTGAAAAGATCTTCAAAGTTGTTTACCCAGTGGATGATCATCATTTCAAGGTT ATTCTCCATTATGGTACACTCGTTATTGACGGTGTGACACCAAACATGATTGACTACTTTGGACGCCCT TACGAGGGAATTGCTGTGTTTGACGGCAAGAAGATCACAGTTACTGGAACTCTGTGGAACGGCAACAAG ATCATTGATGAGCGCCTGATCAACCCAGATGGTTCACTCCTCTTCCGCGTTACTATCAATGGAGTCACC GGATGGCGCCTTTGCGAGCGTATTCTTGCC

21(9F6 protein)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQKLGVSITPIQKIVLSGENGLKIDIHVIIPYEGLSG YQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILA

SEQ ID NO 22(IV opt nucleotide)

atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaaccaagaccaagtc cttgagcagggcggtctgtccagtttgtttcagaaactcggggtgtccgtaacaccgatccaaaagatt gtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc ttccagatgggcctcattgagatgatctttaaggtggtgtaccctgtggatgatcatcactttaaggtg attctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgacc ggctggcggctgtgcgagcgcattttggcg

SEQ ID NO 23(IV optB nucleotides)

atggtattcacactggaggattttgtcggtgattggcggcaaaccgctgggtacaaccaggaccaggtt ctcgaacaagggggcctcagctccctgtttcaaaaactgggtgttagcgttacacctattcaaaaaatc gtgctctccggggaaaacgggctcaaaatcgatattcatgtgattatcccttacgaagggctctccggg tttcagatggggctgatcgaaatgatctttaaggtcgtctatcccgtagatgatcaccacttcaaggtg atcctccactacgggaccctcgtaattgatggcgtgacccccaacatgatcgactattttgggcgccct tacgaggggattgctgtcttcgatggcaaaaaaattacagtgacaggcacactctggaacgggaataag atcattgatgagcgcctgattaatcccgatgggagcctgctctttcgggtgacaattaacggcgtaaca ggctggcgcctctgtgaacggattctggcg

24(9B8 opt nucleotide) SEQ ID NO

atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtc cttgagcagggcggtctgtccagtttgtttcagaaactcggggtgtccgtaacaccgatccaaaagatt gtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc tatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg attctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgacc ggctggcggctgtgcgagcgcattttggcg

SEQ ID NO. 25(9B8 opt B nucleotide)

atggtattcacactggaggattttgtcggtgattggcggcaaaccgctgggtacaacctcgaccaggtt ctcgaacaagggggcctcagctccctgtttcaaaaactgggtgttagcgttacacctattcaaaaaatc gtgctctccggggaaaacgggctcaaaatcgatattcatgtgattatcccttacgaagggctctccggg tatcagatggggcagatcgaaaaaatctttaaggtcgtctatcccgtagatgatcaccacttcaaggtg atcctccactacgggaccctcgtaattgatggcgtgacccccaacatgatcgactattttgggcgccct tacgaggggattgctgtcttcgatggcaaaaaaattacagtgacaggcacactctggaacgggaataag atcattgatgagcgcctgattaatcccgatgggagcctgctctttcgggtgacaattaacggcgtaaca ggctggcgcctctgtgaacggattctggcg

26 (nucleotide 8A 3)

Atggtgattacattggaggatttcgttggagactggcggcagacagctggatacaaccaagatcaagtg ttagaacaaggaggagtgtctagtctgttccaaaagctgggagtgtcaatcaccccaatccagaaaatt gtgctgtctggggagaatgggttaaaaattgatattcatgtcatcatcccttacgagggactcagtggt tttcaaatgggtctgattgaaatgatcttcaaagttgtttacccagtggatgatcatcatttcaaggtt attctccattatggtacactcgttattgacggtgtgacaccaaacatgattgactactttggacgccct tacgagggaattgctgtgtttgacggcaagaagatcacagttactggaactctgtggaacggcaacaag atcattgatgagcgcctgatcaacccagatggttcactcctcttccgcgttactatcaatggagtcacc ggatggcgcctttgcgagcgtattcttgcc

27(8A3 protein)

MVITLEDFVGDWRQTAGYNQDQVLEQGGVSSLFQKLGVSITPIQKIVLSGENGLKIDIHVIIPYEGLSG FQMGLIEMIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILA

SEQ ID NO 28(Luc2 nucleotide)

atggaagatgccaaaaacattaagaagggcccagcgccattctacccactcgaagacgggaccgccggc gagcagctgcacaaagccatgaagcgctacgccctggtgcccggcaccatcgcctttaccgacgcacat atcgaggtggacattacctacgccgagtacttcgagatgagcgttcggctggcagaagctatgaagcgc tatgggctgaatacaaaccatcggatcgtggtgtgcagcgagaatagcttgcagttcttcatgcccgtg ttgggtgccctgttcatcggtgtggctgtggccccagctaacgacatctacaacgagcgcgagctgctg aacagcatgggcatcagccagcccaccgtcgtattcgtgagcaagaaagggctgcaaaagatcctcaac gtgcaaaagaagctaccgatcatacaaaagatcatcatcatggatagcaagaccgactaccagggcttc caaagcatgtacaccttcgtgacttcccatttgccacccggcttcaacgagtacgacttcgtgcccgag agcttcgaccgggacaaaaccatcgccctgatcatgaacagtagtggcagtaccggattgcccaagggc gtagccctaccgcaccgcaccgcttgtgtccgattcagtcatgcccgcgaccccatcttcggcaaccag atcatccccgacaccgctatcctcagcgtggtgccatttcaccacggcttcggcatgttcaccacgctg ggctacttgatctgcggctttcgggtcgtgctcatgtaccgcttcgaggaggagctattcttgcgcagc ttgcaagactataagattcaatctgccctgctggtgcccacactatttagcttcttcgctaagagcact ctcatcgacaagtacgacctaagcaacttgcacgagatcgccagcggcggggcgccgctcagcaaggag gtaggtgaggccgtggccaaacgcttccacctaccaggcatccgccagggctacggcctgacagaaaca accagcgccattctgatcacccccgaaggggacgacaagcctggcgcagtaggcaaggtggtgcccttc ttcgaggctaaggtggtggacttggacaccggtaagacactgggtgtgaaccagcgcggcgagctgtgc gtccgtggccccatgatcatgagcggctacgttaacaaccccgaggctacaaacgctctcatcgacaag gacggctggctgcacagcggcgacatcgcctactgggacgaggacgagcacttcttcatcgtggaccgg ctgaagagcctgatcaaatacaagggctaccaggtagccccagccgaactggagagcatcctgctgcaa caccccaacatcttcgacgccggggtcgccggcctgcccgacgacgatgccggcgagctgcccgccgca gtcgtcgtgctggaacacggtaaaaccatgaccgagaaggagatcgtggactatgtggccagccaggtt acaaccgccaagaagctgcgcggtggtgttgtgttcgtggacgaggtgcctaaaggactgaccggcaag ttggacgcccgcaagatccgcgagattctcattaaggccaagaagggcggcaagatcgccgtt

SEQ ID NO:29(Luc2 protein)

medaknikkgpapfypledgtageqlhkamkryalvpgtiaftdahievdityaeyfemsvrlaeamkr yglntnhrivvcsenslqffmpvlgalfigvavapandiynerellnsmgisqptvvfvskkglqkiln vqkklpiiqkiiimdsktdyqgfqsmytfvtshlppgfneydfvpesfdrdktialimnssgstglpkg valphrtacvrfshardpifgnqiipdtailsvvpfhhgfgmfttlgylicgfrvvlmyrfeeelflrs lqdykiqsallvptlfsffakstlidkydlsnlheiasggaplskevgeavakrfhlpgirqgygltet tsailitpegddkpgavgkvvpffeakvvdldtgktlgvnqrgelcvrgpmimsgyvnnpeatnalidk dgwlhsgdiaywdedehffivdrlkslikykgyqvapaelesillqhpnifdagvaglpdddagelpaa vvvlehgktmtekeivdyvasqvttakklrggvvfvdevpkgltgkldarkireilikakkggkiav

SEQ ID NO 30(HRL (humanized Renilla) nucleotides)

Atggcttccaaggtgtacgaccccgagcaacgcaaacgcatgatcactgggcctcagtggtgggctcgc tgcaagcaaatgaacgtgctggactccttcatcaactactatgattccgagaagcacgccgagaacgcc gtgatttttctgcatggtaacgctgcctccagctacctgtggaggcacgtcgtgcctcacatcgagccc gtggctagatgcatcatccctgatctgatcggaatgggtaagtccggcaagagcgggaatggctcatat cgcctcctggatcactacaagtacctcaccgcttggttcgagctgctgaaccttccaaagaaaatcatc tttgtgggccacgactggggggcttgtctggcctttcactactcctacgagcaccaagacaagatcaag gccatcgtccatgctgagagtgtcgtggacgtgatcgagtcctgggacgagtggcctgacatcgaggag gatatcgccctgatcaagagcgaagagggcgagaaaatggtgcttgagaataacttcttcgtcgagacc atgctcccaagcaagatcatgcggaaactggagcctgaggagttcgctgcctacctggagccattcaag gagaagggcgaggttagacggcctaccctctcctggcctcgcgagatccctctcgttaagggaggcaag cccgacgtcgtccagattgtccgcaactacaacgcctaccttcgggccagcgacgatctgcctaagatg ttcatcgagtccgaccctgggttcttttccaacgctattgtcgagggagctaagaagttccctaacacc gagttcgtgaaggtgaagggcctccacttcagccaggaggacgctccagatgaaatgggtaagtacatc aagagcttcgtggagcgcgtgctgaagaacgagcag

31(HRL (humanized Renilla) protein)

MASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIFLHGNAASSYLWRHVVPHIEP VARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELLNLPKKIIFVGHDWGACLAFHYSYEHQDKIK AIVHAESVVDVIESWDEWPDIEEDIALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEPFK EKGEVRRPTLSWPREIPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNT EFVKVKGLHFSQEDAPDEMGKYIKSFVERVLKNEQ

SEQ ID NO:32(Id-HRL-HT7 sandwich fusion nucleotide)

atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctg gacgagcagcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtg cccaccctgccccagaaccgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagg gacctgcagctggagctgaactcggagtctgaagtcgggaccaccggaggccggggactgcctgtccgc gccccgctcagcaccctgaacggcgagatcagtgccttggcggccgaggcggcatgtgttccagccgac gatcgcatcttgtgtcgcgtttctcttgagaatctttattttcaggcgtctggaggtggtggcggagcg atcgccatggcttccaaggtgtacgaccccgagcaacgcaaacgcatgatcactgggcctcagtggtgg gctcgctgcaagcaaatgaacgtgctggactccttcatcaactactatgattccgagaagcacgccgag aacgccgtgatttttctgcatggtaacgctgcctccagctacctgtggaggcacgtcgtgcctcacatc gagcccgtggctagatgcatcatccctgatctgatcggaatgggtaagtccggcaagagcgggaatggc tcatatcgcctcctggatcactacaagtacctcaccgcttggttcgagctgctgaaccttccaaagaaa atcatctttgtgggccacgactggggggcttgtctggcctttcactactcctacgagcaccaagacaag atcaaggccatcgtccatgctgagagtgtcgtggacgtgatcgagtcctgggacgagtggcctgacatc gaggaggatatcgccctgatcaagagcgaagagggcgagaaaatggtgcttgagaataacttcttcgtc gagaccatgctcccaagcaagatcatgcggaaactggagcctgaggagttcgctgcctacctggagcca ttcaaggagaagggcgaggttagacggcctaccctctcctggcctcgcgagatccctctcgttaaggga ggcaagcccgacgtcgtccagattgtccgcaactacaacgcctaccttcgggccagcgacgatctgcct aagatgttcatcgagtccgaccctgggttcttttccaacgctattgtcgagggagctaagaagttccct aacaccgagttcgtgaaggtgaagggcctccacttcagccaggaggacgctccagatgaaatgggtaag tacatcaagagcttcgtggagcgcgtgctgaagaacgagcaggtttctctcgagccaaccactgaggat ctgtactttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtg gaagtcctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctg cacggtaacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgc attgctccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccac gtccgcttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactgg ggctccgctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggag ttcatccgccctatcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgc accaccgacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggt gtcgtccgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgc gagccactgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtc gaagaatacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgtt ctgatcccaccggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggc ccgggtctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtct actctggagatttccggt

33(Id-HRL-HT7 sandwich fusion protein)

MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIR DLQLELNSESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGA IAMASKVYDPEQRKRMITGPQWWARCKQMNVLDSFINYYDSEKHAENAVIFLHGNAASSYLWRHVVPHI EPVARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELLNLPKKIIFVGHDWGACLAFHYSYEHQDK IKAIVHAESVVDVIESWDEWPDIEEDIALIKSEEGEKMVLENNFFVETMLPSKIMRKLEPEEFAAYLEP FKEKGEVRRPTLSWPREIPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIESDPGFFSNAIVEGAKKFP NTEFVKVKGLHFSQEDAPDEMGKYIKSFVERVLKNEQVSLEPTTEDLYFQSDNDGSEIGTGFPFDPHYV EVLGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDH VRFMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFR TTDVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLS TLEISG

34(8A3-HT7 fusion nucleotide)

atggtgattacattggaggatttcgttggagactggcggcagacagctggatacaaccaagatcaagtg ttagaacaaggaggagtgtctagtctgttccaaaagctgggagtgtcaatcaccccaatccagaaaatt gtgctgtctggggagaatgggttaaaaattgatattcatgtcatcatcccttacgagggactcagtggt tttcaaatgggtctgattgaaatgatcttcaaagttgtttacccagtggatgatcatcatttcaaggtt attctccattatggtacactcgttattgacggtgtgacaccaaacatgattgactactttggacgccct tacgagggaattgctgtgtttgacggcaagaagatcacagttactggaactctgtggaacggcaacaag atcattgatgagcgcctgatcaacccagatggttcactcctcttccgcgttactatcaatggagtcacc ggatggcgcctttgcgagcgtattcttgccggatatctcgagccaaccactgaggatctgtactttcag agcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtcctgggc gagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggtaacccg acctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgctccagac ctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgcttcatg gatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctccgctctg ggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccct atcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtc ggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccg ctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtgg cgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatg gactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatcccaccg gccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggtctgaat ctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctggagatt tccggt

35(8A3-HT7 fusion protein)

MVITLEDFVGDWRQTAGYNQDQVLEQGGVSSLFQKLGVSITPIQKIVLSGENGLKIDIHVIIPYEGLSG FQMGLIEMIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLG ERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFM DAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDV GRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYM DWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEI SG

36(9B8-HT7 fusion nucleotide)

atggtgtttacattggaggatttcgttggagactggcggcagacagctggatacaacctagatcaagtg ttagaacaaggaggattgtctagtctgttccaaaagctgggagtgtcagtcaccccaatccagaaaatt gtgctgtctggggagaatgggttaaaaattgatattcatgtcatcatcccttacgagggactcagtggt tatcaaatgggtcagattgaaaagatcttcaaagttgtttacccagtggatgatcatcatttcaaggtt attctccattatggtacactcgttattgacggtgtgacaccaaacatgattgactactttggacgccct tacgagggaattgctgtgtttgacggcaagaagatcacagttactggaactctgtggaacggcaacaag atcattgatgagcgcctgatcaacccagatggttcactcctcttccgcgttactatcaatggagtcacc ggatggcgcctttgcgagcgtattcttgccggatatctcgagccaaccactgaggatctgtactttcag agcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtcctgggc gagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggtaacccg acctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgctccagac ctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgcttcatg gatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctccgctctg ggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccct atcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtc ggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccg ctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtgg cgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatg gactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatcccaccg gccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggtctgaat ctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctggagatt tccggt

37(9B8-HT7 fusion protein)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSG YQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLG ERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFM DAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDV GRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYM DWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEI SG

38(9F6-HT7 fusion nucleotide)

atggtgtttacattggaggatttcgttggagactggcggcagacagctggatacaacctagatcaagtg ttagaacaaggaggagtgtctagtctgttccaaaagctgggagtgtcaatcaccccaatccagaaaatt gtgctgtctggggagaatgggttaaaaattgatattcatgtcatcatcccttacgagggactcagtggt tatcaaatgggtcagattgaaaagatcttcaaagttgtttacccagtggatgatcatcatttcaaggtt attctccattatggtacactcgttattgacggtgtgacaccaaacatgattgactactttggacgccct tacgagggaattgctgtgtttgacggcaagaagatcacagttactggaactctgtggaacggcaacaag atcattgatgagcgcctgatcaacccagatggttcactcctcttccgcgttactatcaatggagtcacc ggatggcgcctttgcgagcgtattcttgccggatatctcgagccaaccactgaggatctgtactttcag agcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtcctgggc gagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggtaacccg acctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgctccagac ctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgcttcatg gatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctccgctctg ggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccct atcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtc ggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccg ctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtgg cgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatg gactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatcccaccg gccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggtctgaat ctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctggagatt tccggt

39(9F6-HT7 fusion protein)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQKLGVSITPIQKIVLSGENGLKIDIHVIIPYEGLSG YQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLG ERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFM DAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDV GRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYM DWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEI SG

40(Id-9B8 opt-HT7 sandwich fusion nucleotide) SEQ ID NO

atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctg gacgagcagcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtg cccaccctgccccagaaccgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagg gacctgcagctggagctgaactcggagtctgaagtcgggaccaccggaggccggggactgcctgtccgc gccccgctcagcaccctgaacggcgagatcagtgccttggcggccgaggcggcatgtgttccagccgac gatcgcatcttgtgtcgcgtttctcttgagaatctttattttcaggcgtctggaggtggtggcggagcg atcgccatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggac caagtccttgagcagggcggtctgtccagtttgtttcagaaactcggggtgtccgtaacaccgatccaa aagattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctg agcggctatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcacttt aaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggc aacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacgga gtgaccggctggcggctgtgcgagcgtattcttgccggatatctcgagccaaccactgaggatctgtac tttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtc ctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggt aacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgct ccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgc ttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctcc gctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatc cgccctatcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccacc gacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtc cgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagcca ctgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaa tacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatc ccaccggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggt ctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctg gagatttccggtt

41(Id-9B8 opt-HT7 sandwich fusion protein)

MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIR DLQLELNSESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGA IAMVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGL SGYQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNG NKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEV LGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVR FMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTT DVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTL EISG

42(9B8 opt + K33N nucleotides)

Atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtc cttgagcagggcggtctgtccagtttgtttcagaatctcggggtgtccgtaacaccgatccaaaagatt gtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc tatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg attctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgacc ggctggcggctgtgcgagcgcattttggcg

43(9B8 opt + K33N protein) SEQ ID NO

MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSG YQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILA

44(QC27-HT7 nucleotide) SEQ ID NO

atggtgtttacattggaggatttcgttggagactggcggcagacagctggatacaacctagatcaagtg ttagaacaaggaggattgtctagtctgttccaaaagctgggagtgtcagtcaccccaatccagaaaatt gtgctgtctggggagaatgggttaaaaattgatattcatgtcatcatcccttacgagggactcagtggt tttcaaatgggtctgattgaaatgatcttcaaagttgtttacccagtggatgatcatcatttcaagatt attcaccattatggtacactcgttattgacggtgtgacaccaaacatgattgacttctttggacgccct tacgagggaattgctgtgtttgacggcaagaagatcacagttactggaactctgtggaacggcaacaag atcattgatgagcgcctgatcaacccagatggttcactcctcttccgcgttactatcaatggagtcacc ggatggcgcctttgcgagcgtattcttgccggatatctcgagccaaccactgaggatctgtactttcag agcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtcctgggc gagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggtaacccg acctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgctccagac ctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgcttcatg gatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctccgctctg ggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccct atcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtc ggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccg ctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtgg cgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatg gactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatcccaccg gccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggtctgaat ctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctggagatt tccggt

SEQ ID NO:45(QC27-HT7 protein)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSG FQMGLIEMIFKVVYPVDDHHFKIIHHYGTLVIDGVTPNMIDFFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLG ERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFM DAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDV GRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYM DWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEI SG

SEQ ID NO:46(8F2)

atggtgtttacattggaggatttcgttggagactggcggcagacagctggatacaaccaagatcaagtg ttagaacaaggaggagtgtctagtctgttccaaaagctgggagtgtcagtcaccccaatccagaaaatt gtgctgtctggggagaatgggttaaaaattgatattcatgtcatcatcccttacgagggactcagtggt tttcaaatgggtctgattgaaatgatcttcaaagttgtttacccagtggatgatcatcatttcaaggtt attctccattatggtacactcgttattgacggtgtgacaccaaacatgattgactactttggacgccct tacgagggaattgctgtgtttgacggcaagaagatcacagttactggaactctgtggaacggcaacaag atcattgatgagcgcctgatcaacccagatggttcactcctcttccgcgttactatcaatggagtcacc ggatggcgcctttgcgagcgtattcttgcc

47(8F2 protein)

MVFTLEDFVGDWRQTAGYNQDQVLEQGGVSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSG FQMGLIEMIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILA

SEQ ID NO 48(IV-HT7 nucleotide)

atggtgtttacattggaggatttcgttggagactggcggcagacagctggatacaaccaagatcaagtg ttagaacaaggaggattgtctagtctgttccaaaagctgggagtgtcagtcaccccaatccagaaaatt gtgctgtctggggagaatgggttaaaaattgatattcatgtcatcatcccttacgagggactcagtggt tttcaaatgggtctgattgaaatgatcttcaaagttgtttacccagtggatgatcatcatttcaaggtt attctccattatggtacactcgttattgacggtgtgacaccaaacatgattgactactttggacgccct tacgagggaattgctgtgtttgacggcaagaagatcacagttactggaactctgtggaacggcaacaag atcattgatgagcgcctgatcaacccagatggttcactcctcttccgcgttactatcaatggagtcacc ggatggcgcctttgcgagcgtattcttgccggatatctcgagccaaccactgaggatctgtactttcag agcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtcctgggc gagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggtaacccg acctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgctccagac ctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgcttcatg gatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctccgctctg ggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccct atcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtc ggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccg ctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtgg cgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatg gactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatcccaccg gccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggtctgaat ctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctggagatt tccggt

49(IV-HT7 protein)

MVFTLEDFVGDWRQTAGYNQDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSG FQMGLIEMIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLG ERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFM DAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDV GRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYM DWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEI SG

50(8F2-HT7 nucleotide) SEQ ID NO

atggtgtttacattggaggatttcgttggagactggcggcagacagctggatacaaccaagatcaagtg ttagaacaaggaggagtgtctagtctgttccaaaagctgggagtgtcagtcaccccaatccagaaaatt gtgctgtctggggagaatgggttaaaaattgatattcatgtcatcatcccttacgagggactcagtggt tttcaaatgggtctgattgaaatgatcttcaaagttgtttacccagtggatgatcatcatttcaaggtt attctccattatggtacactcgttattgacggtgtgacaccaaacatgattgactactttggacgccct tacgagggaattgctgtgtttgacggcaagaagatcacagttactggaactctgtggaacggcaacaag atcattgatgagcgcctgatcaacccagatggttcactcctcttccgcgttactatcaatggagtcacc ggatggcgcctttgcgagcgtattcttgccggatatctcgagccaaccactgaggatctgtactttcag agcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtcctgggc gagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggtaacccg acctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgctccagac ctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgcttcatg gatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctccgctctg ggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccct atcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtc ggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccg ctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtgg cgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatg gactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatcccaccg gccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggtctgaat ctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctggagatt tccggt

51(8F2-HT7 protein)

MVFTLEDFVGDWRQTAGYNQDQVLEQGGVSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSG FQMGLIEMIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLG ERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFM DAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDV GRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYM DWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEI SG

52(15C1-HT7 nucleotide)

atggtgtttacattgaaggatttcgttggagactggcggcagacagctggatacaaccaagatcaagtg ttagaacaaggaggattgtctagtctgttccaaaatctgggagtgtcagtcaccccaatccagaaaatt gtgctgtctggggagaatgggttaaaaattgatattcatgtcatcatcccttacgagggactcagtggt tatcaaatgggtcagattgaaaagatcttcaaagttgtttacccagtggatgatcatcatttcaaggtt attctccattatggtacactcgttattgacggtgtgacaccaaacatgattgactactttggacgccct tacgagggaattgctgtgtttgacggcaagaagatcacagttactggaactctgtggaacggcaacaag atcattgatgagcgcctgatcaacccagatggttcactcctcttccgcgttactatcaatggagtcacc ggatggcgcctttgcgagcgtattcttgccggatatctcgagccaaccactgaggatctgtactttcag agcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtcctgggc gagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggtaacccg acctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgctccagac ctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgcttcatg gatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctccgctctg ggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccct atcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtc ggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccg ctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtgg cgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatg gactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatcccaccg gccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggtctgaat ctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctggagatt tccggt

53(15C1-HT7 protein)

MVFTLKDFVGDWRQTAGYNQDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSG YQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEVLG ERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFM DAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDV GRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEEYM DWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTLEI SG

54(OgLuc secretion signal peptide) Cereus tenuis (SEQ ID NO: 54)

atggcttactccacactgttcatcattgctctcacagccgtcgtaacacaagcctcctccacacagaaa agcaacctgaca

55(Id-C1A4E-HT7 sandwich fusion nucleotide)

atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctg gacgagcagcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtg cccaccctgccccagaaccgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagg gacctgcagctggagctgaactcggagtctgaagtcgggaccaccggaggccggggactgcctgtccgc gccccgctcagcaccctgaacggcgagatcagtgccttggcggccgaggcggcatgtgttccagccgac gatcgcatcttgtgtcgcgtttctcttgagaatctttattttcaggcgtctggaggtggtggcggagcg atcgccatggtgtttacattggaggatttcgttggagactggcggcagacagctggatacaaccaagat caagtgttagaacaaggaggattgtctagtctgttccaaaagctgggagtgtcagtcaccccaatccag aaaattgtgctgtctggggagaatgggttaaaatttgatattcatgtcatcatcccttacgagggactc agtggttttcaaatgggtctgattgaaatgatcttcaaagttgtttacccagtggatgatcatcatttc aagattattctccattatggtacactcgttattgacggtgtgacaccaaacatgattgactactttgga cgcccttacgagggaattgctgtgtttgacggcaagaagatcacagttactggaactctgtggaacggc aacaagatcattgatgagcgcctgatcaacccagatggttcactcctcttccgcgttactatcaatgga gtcaccggatggcgcctttgcgagcgtattcttgccggatctctcgagccaaccactgaggatctgtac tttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtc ctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggt aacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgct ccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgc ttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctcc gctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatc cgccctatcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccacc gacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtc cgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagcca ctgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaa tacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatc ccaccggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggt ctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctg gagatttccggt

56(Id-C1A4E-HT7 sandwich fusion protein)

MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIR DLQLELNSESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGA IAMVFTLEDFVGDWRQTAGYNQDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKFDIHVIIPYEGL SGFQMGLIEMIFKVVYPVDDHHFKIILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNG NKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEV LGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVR FMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTT DVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTL EISG

57(Id-IV-HT7 sandwich fusion nucleotide) SEQ ID NO

atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctg gacgagcagcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtg cccaccctgccccagaaccgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagg gacctgcagctggagctgaactcggagtctgaagtcgggaccaccggaggccggggactgcctgtccgc gccccgctcagcaccctgaacggcgagatcagtgccttggcggccgaggcggcatgtgttccagccgac gatcgcatcttgtgtcgcgtttctcttgagaatctttattttcaggcgtctggaggtggtggcggagcg atcgccatggtgtttacattggaggatttcgttggagactggcggcagacagctggatacaaccaagat caagtgttagaacaaggaggattgtctagtctgttccaaaagctgggagtgtcagtcaccccaatccag aaaattgtgctgtctggggagaatgggttaaaaattgatattcatgtcatcatcccttacgagggactc agtggttttcaaatgggtctgattgaaatgatcttcaaagttgtttacccagtggatgatcatcatttc aaggttattctccattatggtacactcgttattgacggtgtgacaccaaacatgattgactactttgga cgcccttacgagggaattgctgtgtttgacggcaagaagatcacagttactggaactctgtggaacggc aacaagatcattgatgagcgcctgatcaacccagatggttcactcctcttccgcgttactatcaatgga gtcaccggatggcgcctttgcgagcgtattcttgccgtttctctcgagccaaccactgaggatctgtac tttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtc ctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggt aacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgct ccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgc ttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctcc gctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatc cgccctatcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccacc gacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtc cgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagcca ctgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaa tacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatc ccaccggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggt ctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctg gagatttccggt

58(Id-IV-HT7 sandwich fusion protein)

MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIR DLQLELNSESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGA IAMVFTLEDFVGDWRQTAGYNQDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGL SGFQMGLIEMIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNG NKIIDERLINPDGSLLFRVTINGVTGWRLCERILAVSLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEV LGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVR FMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTT DVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTL EISG

59(Id-9B8 opt + K33N-HT7 sandwich fusion nucleotide)

atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctg gacgagcagcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtg cccaccctgccccagaaccgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagg gacctgcagctggagctgaactcggagtctgaagtcgggaccaccggaggccggggactgcctgtccgc gccccgctcagcaccctgaacggcgagatcagtgccttggcggccgaggcggcatgtgttccagccgac gatcgcatcttgtgtcgcgtttctcttgagaatctttattttcaggcgtctggaggtggtggcggagcg atcgccatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggac caagtccttgagcagggcggtctgtccagtttgtttcagaatctcggggtgtccgtaacaccgatccaa aagattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctg agcggctatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcacttt aaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggc aacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacgga gtgaccggctggcggctgtgcgagcgcattttggcgggatatctcgagccaaccactgaggatctgtac tttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtc ctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggt aacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgct ccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgc ttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctcc gctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatc cgccctatcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccacc gacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtc cgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagcca ctgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaa tacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatc ccaccggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggt ctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctg gagatttccggt

60(Id-9B8 opt + K33N-HT7 sandwich fusion protein)

MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIR DLQLELNSESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGA IAMVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGL SGYQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNG NKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEV LGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVR FMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTT DVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTL EISG

61(Id-9B8-HT7 sandwich fusion nucleotide)

atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctg gacgagcagcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtg cccaccctgccccagaaccgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagg gacctgcagctggagctgaactcggagtctgaagtcgggaccaccggaggccggggactgcctgtccgc gccccgctcagcaccctgaacggcgagatcagtgccttggcggccgaggcggcatgtgttccagccgac gatcgcatcttgtgtcgcgtttctcttgagaatctttattttcaggcgtctggaggtggtggcggagcg atcgccatggtgtttacattggaggatttcgttggagactggcggcagacagctggatacaacctagat caagtgttagaacaaggaggattgtctagtctgttccaaaagctgggagtgtcagtcaccccaatccag aaaattgtgctgtctggggagaatgggttaaaaattgatattcatgtcatcatcccttacgagggactc agtggttatcaaatgggtcagattgaaaagatcttcaaagttgtttacccagtggatgatcatcatttc aaggttattctccattatggtacactcgttattgacggtgtgacaccaaacatgattgactactttgga cgcccttacgagggaattgctgtgtttgacggcaagaagatcacagttactggaactctgtggaacggc aacaagatcattgatgagcgcctgatcaacccagatggttcactcctcttccgcgttactatcaatgga gtcaccggatggcgcctttgcgagcgtattcttgccgtttctctcgagccaaccactgaggatctgtac tttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtc ctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggt aacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgct ccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgc ttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctcc gctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatc cgccctatcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccacc gacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtc cgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagcca ctgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaa tacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatc ccaccggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggt ctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctg gagatttccggt

62(Id-9B8-HT7 sandwich fusion protein)

MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIR DLQLELNSESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGA IAMVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGL SGYQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNG NKIIDERLINPDGSLLFRVTINGVTGWRLCERILAVSLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEV LGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVR FMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTT DVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTL EISG

63(Id-9F6-HT7 sandwich fusion nucleotide)

atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctg gacgagcagcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtg cccaccctgccccagaaccgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagg gacctgcagctggagctgaactcggagtctgaagtcgggaccaccggaggccggggactgcctgtccgc gccccgctcagcaccctgaacggcgagatcagtgccttggcggccgaggcggcatgtgttccagccgac gatcgcatcttgtgtcgcgtttctcttgagaatctttattttcaggcgtctggaggtggtggcggagcg atcgccatggtgtttacattggaggatttcgttggagactggcggcagacagctggatacaacctagat caagtgttagaacaaggaggagtgtctagtctgttccaaaagctgggagtgtcaatcaccccaatccag aaaattgtgctgtctggggagaatgggttaaaaattgatattcatgtcatcatcccttacgagggactc agtggttatcaaatgggtcagattgaaaagatcttcaaagttgtttacccagtggatgatcatcatttc aaggttattctccattatggtacactcgttattgacggtgtgacaccaaacatgattgactactttgga cgcccttacgagggaattgctgtgtttgacggcaagaagatcacagttactggaactctgtggaacggc aacaagatcattgatgagcgcctgatcaacccagatggttcactcctcttccgcgttactatcaatgga gtcaccggatggcgcctttgcgagcgtattcttgccgtttctctcgagccaaccactgaggatctgtac tttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtc ctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggt aacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgct ccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgc ttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctcc gctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatc cgccctatcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccacc gacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtc cgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagcca ctgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaa tacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatc ccaccggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggt ctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctg gagatttccggt

64(Id-9F6-HT7 sandwich fusion protein)

MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIR DLQLELNSESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGA IAMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQKLGVSITPIQKIVLSGENGLKIDIHVIIPYEGL SGYQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNG NKIIDERLINPDGSLLFRVTINGVTGWRLCERILAVSLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEV LGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVR FMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTT DVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTL EISG

65(9B8 opt-P nucleotide) SEQ ID NO

atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtc cttgagcagggcggtctgtccagtttgtttcagaaactcggggtgtccgtaacaccgatccaaaagatt gtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc tatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg attctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgacc ggctggcggctgtgcgagcgcattttggcgaattctcacggcttccctcccgaggtggaggagcaggcc gccggcaccctgcccatgagctgcgcccaggagagcggcatggatagacaccctgctgcttgcgccagc gccaggatcaacgtc

SEQ ID NO:66(2X ARE)

TAGCTTGGAAATGACATTGCTAATGGTGACAAAGCAACTTTTAGCTTGGAAATGACATTGCTAATGGTG ACAAAGCAACTTT

SEQ ID NO:67(HRE)

CTGGAATTTTCTAGACTGGAATTTTCTAGACTGGAATTTTCTAGA

68(K33N +170G nucleotide) SEQ ID NO

atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtc cttgagcagggcggtctgtccagtttgtttcagaatctcggggtgtccgtaacaccgatccaaaagatt gtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc tatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg attctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgacc ggctggcggctgtgcgagcgcattttggcggga

69(K33N +170G protein)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSG YQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILAG

70(27A5(NF) nucleotide) SEQ ID NO

ATGGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTACAACCTGGACCAAGTC CTTGAGCAGGGCGGTCTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAAGATT GTCCTGAGCGGTGAAAACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGC TATCAGATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTG ATTCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCG TATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACCGGGACCCTGTGGAACGGCAACAAA ATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGCGTAACCATCAACGGAGTGACC GGCTGGCGGCTGTGCGAGCGCATTTTGGCGGGA

71(27A5(NF) protein)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSG YQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILAG

72(23D4(NF) nucleotide) SEQ ID NO

ATGGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTACAACCTGGACCAAGTC CTTGAGCAGGGCGGCCTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACACCGATCCAAAAGATT GTCCTGAGCGGTGAAAACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGC TATCAGATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTG ATTCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACTTGATCGACTATTTCGGACGTCCG TATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACCGGGACCCTGTGGAACGGCAACAAA ATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGCGTAACCATCAACGGAGTGACC GGCTGGCGGCTGTGCGAGCGCATTTTGGCGGGA

73(23D4(NF) protein)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSG YQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNLIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILAG

74(24C2(NF) nucleotide) SEQ ID NO

ATGGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCAGCAGACAGCCGGCTACAACCTGGACCAAGTC CTTGAGCAGGGCGGTCTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAAGATT GTCCTGAGCGGTGAAAACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGC TATCAGATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTG ATTCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCG TATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACCGGGACCCTGTGGAACGGCAACAAA ATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGCGTAACCATCAACGGAGTGACC GGCTGGCGGCTGTGCGAGCGCATTTTGGCGGGA

75(24C2(NF) protein)

MVFTLEDFVGDWQQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLSG YQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILAG

76(Id-23D4-HT7 sandwich fusion nucleotide)

atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctg gacgagcagcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtg cccaccctgccccagaaccgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagg gacctgcagctggagctgaactcggagtctgaagtcgggaccaccggaggccggggactgcctgtccgc gccccgctcagcaccctgaacggcgagatcagtgccttggcggccgaggcggcatgtgttccagccgac gatcgcatcttgtgtcgcgtttctcttgagaatctttattttcaggcgtctggaggtggtggcggagcg atcgccatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggac caagtccttgagcagggcggcctgtccagtttgtttcagaatctcggggtgtccgtaacaccgatccaa aagattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctg agcggctatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcacttt aaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacttgatcgactatttcgga cgtccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggc aacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacgga gtgaccggctggcggctgtgcgagcgcattttggcgggatatctcgagccaaccactgaggatctgtac tttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtc ctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggt aacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgct ccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgc ttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctcc gctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatc cgccctatcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccacc gacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtc cgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagcca ctgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaa tacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatc ccaccggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggt ctg

77(Id-23D4-HT7 sandwich fusion protein)

MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIR DLQLELNSESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGA IAMVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGL SGYQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNLIDYFGRPYEGIAVFDGKKITVTGTLWNG NKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEV LGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVR FMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTT DVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGL

78(Id-24C2-HT7 sandwich fusion nucleotide)

atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctg gacgagcagcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtg cccaccctgccccagaaccgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagg gacctgcagctggagctgaactcggagtctgaagtcgggaccaccggaggccggggactgcctgtccgc gccccgctcagcaccctgaacggcgagatcagtgccttggcggccgaggcggcatgtgttccagccgac gatcgcatcttgtgtcgcgtttctcttgagaatctttattttcaggcgtctggaggtggtggcggagcg atcgccatggtgtttacactcgaagatttcgtaggggactggcagcagacagccggctacaacctggac caagtccttgagcagggcggtctgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaa aagattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctg agcggctatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcacttt aaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggc aacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacgga gtgaccggctggcggctgtgcgagcgcattttggcgggatatctcgagccaaccactgaggatctgtac tttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtc ctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggt aacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgct ccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgc ttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctcc gctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatc cgccctatcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccacc gacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtc cgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagcca ctgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaa tacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatc ccaccggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggt ctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctg gagatttccggt

79(Id-24C2-HT7 sandwich fusion protein)

MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIR DLQLELNSESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGA IAMVFTLEDFVGDWQQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGL SGYQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNG NKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEV LGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVR FMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTT DVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTL EISG

SEQ ID NO 80(1F7(NF) nucleotide)

ATGGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTACAACCTGGACCAAGTC CTTGAGCAGGGCGGTCTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACACCGATCCAAAGGATT GTCCTGAGCGGTGAAAACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGC GATCAGATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTG ATTCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCG TATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACCGGGACCCTGTGGAACGGCAACAAA ATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGCGTAACCATCAACGGAGTGACC GGCTGGCGGCTGTGCGAGCGCATTTTGGCGGGA

81(1F7(NF) protein)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILAG

82(15H1(NF) nucleotides) SEQ ID NO

ATGGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTACAACCTGGATCAAGTC CTTGAGCAGGGCGGTCTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACACCGATCCAAAAGATT GTCCTGAGCGGTGAAAACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAACGGC TATCAGATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTG ATTCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCG TATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACCGGGACCCTGTGGAACGGCAACAAA ATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGCGTAACCATCAACGGAGTGACC GGCTGGCGGCTGTGCGAGCGCATTTTGGCGGGA

83(15H1(NF) protein)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGLNG YQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILAG

84(Id-1F7-HT7 sandwich fusion nucleotide)

atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctg gacgagcagcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtg cccaccctgccccagaaccgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagg gacctgcagctggagctgaactcggagtctgaagtcgggaccaccggaggccggggactgcctgtccgc gccccgctcagcaccctgaacggcgagatcagtgccttggcggccgaggcggcatgtgttccagccgac gatcgcatcttgtgtcgcgtttctcttgagaatctttattttcaggcgtctggaggtggtggcggagcg atcgccatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggac caagtccttgagcagggcggtctgtccagtttgtttcagaatctcggggtgtccgtaacaccgatccaa aggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctg agcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcacttt aaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggc aacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacgga gtgaccggctggcggctgtgcgagcgcattttggcgggatatctcgagccaaccactgaggatctgtac tttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtc ctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggt aacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgct ccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgc ttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctcc gctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatc cgccctatcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccacc gacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtc cgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagcca ctgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaa tacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatc ccaccggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggt ctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctg gag

85(Id-1F7-HT7 sandwich fusion protein)

MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIR DLQLELNSESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGA IAMVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGL SGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNG NKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEV LGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVR FMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTT DVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTL EISG

86(Id-15H1-HT7 sandwich fusion nucleotide)

atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctg gacgagcagcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtg cccaccctgccccagaaccgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagg gacctgcagctggagctgaactcggagtctgaagtcgggaccaccggaggccggggactgcctgtccgc gccccgctcagcaccctgaacggcgagatcagtgccttggcggccgaggcggcatgtgttccagccgac gatcgcatcttgtgtcgcgtttctcttgagaatctttattttcaggcgtctggaggtggtggcggagcg atcgccatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggat caagtccttgagcagggcggtctgtccagtttgtttcagaatctcggggtgtccgtaacaccgatccaa aagattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctg aacggctatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcacttt aaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggc aacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacgga gtgaccggctggcggctgtgcgagcgcattttggcgggatatctcgagccaaccactgaggatctgtac tttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtc ctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggt aacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgct ccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgc ttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctcc gctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatc cgccctatcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccacc gacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtc cgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagcca ctgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaa tacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatc ccaccggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggt ctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctg gagatttccggt

87(Id-15H1-HT7 sandwich fusion protein)

MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIR DLQLELNSESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGA IAMVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQKIVLSGENGLKIDIHVIIPYEGL NGYQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNG NKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEV LGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVR FMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTT DVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTL EISG

88(9B8 opt + K33N + L27V + T39T + K43R + Y68D nucleotides)

Atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtc cttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg attctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgacc ggctggcggctgtgcgagcgcattttggcg

89(9B8 opt + K33N + L27V + T39T + K43R + Y68D protein)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILA

SEQ ID NO:90(9B8 opt + K33N + L27V + T39T + K43R + Y68D Sanming Zhi fusion nucleotide)

atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctg gacgagcagcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtg cccaccctgccccagaaccgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagg gacctgcagctggagctgaactcggagtctgaagtcgggaccaccggaggccggggactgcctgtccgc gccccgctcagcaccctgaacggcgagatcagtgccttggcggccgaggcggcatgtgttccagccgac gatcgcatcttgtgtcgcgtttctcttgagaatctttattttcaggcgtctggaggtggtggcggagcg atcgccatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggac caagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaa aggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctg agcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcacttt aaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggc aacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacgga gtgaccggctggcggctgtgcgagcgcattttggcgggatatctcgagccaaccactgaggatctgtac tttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtc ctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggt aacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgct ccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgc ttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctcc gctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatc cgccctatcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccacc gacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtc cgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagcca ctgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaa tacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatc ccaccggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggt ctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctg gagatttccggt

91(9B8 opt + K33N + L27V + T39T + K43R + Y68D sandwich fusion protein)

MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIR DLQLELNSESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGA IAMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGL SGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNG NKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEV LGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVR FMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTT DVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTL EISG

92(9B8 opt + K33N + T39T + K43R + Y68D nucleotides)

Atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtc cttgagcagggcggtctgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg attctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgacc ggctggcggctgtgcgagcgcattttggcg

93(9B8 opt + K33N + T39T + K43R + Y68D protein)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILA

94(9B8 opt + K33N + T39T + K43R + Y68D sandwich fusion nucleotide)

atgcttggtctgtcggagcaaagcgtgtccatctcgcgctgcgctgggacgcgcctgcccgccttgctg gacgagcagcaggtgaacgtcctgctctacgacatgaacggctgctactcacgcctcaaggagctggtg cccaccctgccccagaaccgcaaagtgagcaaggtggagatcctgcagcatgtaatcgactacatcagg gacctgcagctggagctgaactcggagtctgaagtcgggaccaccggaggccggggactgcctgtccgc gccccgctcagcaccctgaacggcgagatcagtgccttggcggccgaggcggcatgtgttccagccgac gatcgcatcttgtgtcgcgtttctcttgagaatctttattttcaggcgtctggaggtggtggcggagcg atcgccatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggac caagtccttgagcagggcggtctgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaa aggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctg agcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcacttt aaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggc aacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacgga gtgaccggctggcggctgtgcgagcgcattttggcgggatatctcgagccaaccactgaggatctgtac tttcagagcgataacgatggatccgaaatcggtactggctttccattcgacccccattatgtggaagtc ctgggcgagcgcatgcactacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggt aacccgacctcctcctacgtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgct ccagacctgatcggtatgggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgc ttcatggatgccttcatcgaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctcc gctctgggtttccactgggccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatc cgccctatcccgacctgggacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccacc gacgtcggccgcaagctgatcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtc cgcccgctgactgaagtcgagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagcca ctgtggcgcttcccaaacgagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaa tacatggactggctgcaccagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatc ccaccggccgaagccgctcgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggt ctgaatctgctgcaagaagacaacccggacctgatcggcagcgagatcgcgcgctggctgtctactctg gagatttccggt

SEQ ID NO 95(9B8 opt + K33N + T39T + K43R + Y68D sandwich fusion protein)

MLGLSEQSVSISRCAGTRLPALLDEQQVNVLLYDMNGCYSRLKELVPTLPQNRKVSKVEILQHVIDYIR DLQLELNSESEVGTTGGRGLPVRAPLSTLNGEISALAAEAACVPADDRILCRVSLENLYFQASGGGGGA IAMVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGL SGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNG NKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGYLEPTTEDLYFQSDNDGSEIGTGFPFDPHYVEV LGERMHYVDVGPRDGTPVLFLHGNPTSSYVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVR FMDAFIEALGLEEVVLVIHDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTT DVGRKLIIDQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIGSEIARWLSTL EISG

SEQ ID NO:96(CRE)

GCACCAGACAGTGACGTCAGCTGCCAGATCCCATGGCCGTCATACTGTGACGTCTTTCAGACACCCCTT GACGTCAATGGGAGAACA

97 (nucleotide CP 84 NO linker)

Atggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacc gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgatggtgtttacactc gaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggt gtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaa aacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccag atcgaaaaaatttttaaggtggtgtaccctgtg

98 (protein CP 84 jointless)

MDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLF RVTINGVTGWRLCERILAMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGE NGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPV

99 (nucleotide CP 845 AA linker)

Atggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacc gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcggggagctccggtgga atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtc cttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtg

100 (protein CP 845 AA linker)

MDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLF RVTINGVTGWRLCERILAGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPV

SEQ ID NO 101 (nucleotide CP 8410 AA linker)

Atggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacc gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtgga gggagctccggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctac aacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaact ccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtat gaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtg

SEQ ID NO:102 (protein CP 8410 AA linker)

MDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLF RVTINGVTGWRLCERILAGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVT PIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPV

103 (nucleotide CP 8420 AA linker)

Atggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacc gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtgga gggagctccggtggaggaagttctggtggagggagctccggtggaatggtgtttacactcgaagatttc gtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagt ttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctg aagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaa atttttaaggtggtgtaccctgtg

104 (protein CP 8420 AA linker)

MDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLF RVTINGVTGWRLCERILAGSSGGGSSGGGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSS LFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPV

105 (nucleotide CP 95 NO linker)

Atgggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggc atcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgac gagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcgg ctgtgcgagcgcattttggcgatggtgtttacactcgaagatttcgtaggggactggcggcagacagcc ggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtcc gtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatc ccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtg gatgatcatcactttaaggtgattctgcactat

106 (protein CP 95 jointless)

MGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR LCERILAMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVII PYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHY

107 (nucleotide CP 955 AA linker) SEQ ID NO

Atgggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggc atcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgac gagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcgg ctgtgcgagcgcattttggcggggagctccggtggaatggtgtttacactcgaagatttcgtaggggac tggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcag aatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgac atccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaag gtggtgtaccctgtggatgatcatcactttaaggtgattctgcactat

108 (protein CP 955 AA linker)

MGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR LCERILAGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKID IHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHY

109 (nucleotide CP 9510 AA linker)

Atgggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggc atcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgac gagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcgg ctgtgcgagcgcattttggcgggaagttctggtggagggagctccggtggaatggtgtttacactcgaa gatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtg tccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaac ggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatc gaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactat

110 (protein CP 9510 AA linker)

MGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR LCERILAGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGEN GLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHY

111 (nucleotide CP 9520 AA linker)

Atgggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggc atcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgac gagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcgg ctgtgcgagcgcattttggcgggaagttctggtggagggagctccggtggaggaagttctggtggaggg agctccggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaac ctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccg atccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaa ggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcat cactttaaggtgattctgcactat

SEQ ID NO:112 (protein CP 9520 AA linker)

MGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR LCERILAGSSGGGSSGGGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTP IQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHY

113 (Joint) SEQ ID NO

(GSGG)n

SEQ ID NO:114(L27V CP 5 TEV)

Atggatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggt gtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaa aacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccag atcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggc acactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgcc gtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgc ctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgc gagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtac ttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaa

SEQ ID NO:115(L27V CP 5 TEV)

MDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQ IEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDER LINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLE

SEQ ID NO:116(L27V CP 6 TEV)

atgttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtg tccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaac ggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatc gaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcaca ctggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtg ttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctg atcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgag cgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttc cagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagat

SEQ ID NO:117(L27V CP 6 TEV)

MFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQI EKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERL INPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLED

SEQ ID NO:118(L27V CP 7 TEV)

atggtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtcc agtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggc ctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaa aaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactg gtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttc gacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatc aaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgc attttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccag agcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttc

SEQ ID NO:119(L27V CP 7 TEV)

MVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIE KIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLI NPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDF

SEQ ID NO:120(L27V CP 9 TEV)

atggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttg tttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaag atcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatt tttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatc gacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggc aaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacccc gacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttg gcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgat aacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggg

SEQ ID NO:121(L27V CP 9 TEV)

MDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKI FKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINP DGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVG

SEQ ID NO:122(L27V CP 11 TEV)

Atgcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcag aatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgac atccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaag gtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggg gttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaag atcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggc tccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcggga agttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacgga agttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactgg

SEQ ID NO:123(L27V CP 11 TEV)

MRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFK VVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDG SLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDW

SEQ ID NO:124(L27V CP 12 TEV)

Cagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccat gtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtg taccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacg ccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcact gtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctg ctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttct ggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttct ggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggg

SEQ ID NO:125(L27V CP 12 TEV)

QTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVV YPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSL LFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVG

SEQ ID NO:126(L27V CP 15 TEV)

atgggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtg tccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatc atcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccct gtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacc gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtgga ggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtgga ggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagcc

SEQ ID NO:127(L27V CP 15 TEV)

MGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYP VDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLF RVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTA

SEQ ID NO:128(L27V CP 18 TEV)

ctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccg atccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaa ggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcat cactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactat ttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtgg aacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatc aacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggt ggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggt ggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaac

SEQ ID NO:129(L27V CP 18 TEV)

LDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDH HFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTI NGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYN

SEQ ID NO:130(L27V CP 21 TEV)

gtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaagg attgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagc ggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaag gtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacgg ccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaac aaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtg accggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcct actactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtg tttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaa

SEQ ID NO:131(L27V CP 21 TEV)

VLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFK VILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGV TGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQ

SEQ ID NO:132(L27V CP 24 TEV)

Cagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctg agcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcag atgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctg cactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaa ggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatc gacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctgg cggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgag aacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactc gaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgag

SEQ ID NO:133(L27V CP 24 TEV)

QGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVIL HYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW RLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLE

SEQ ID NO:134(L27V CP 27 TEV)

gtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaa aacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccag atcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggc acactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgcc gtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgc ctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgc gagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtac ttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttc gtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggt

SEQ ID NO:135(L27V CP 27 TEV)

VSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYG TLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLC ERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGG

SEQ ID NO:136(L27V CP 34 TEV)

atgctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgac atccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaag gtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggg gttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaag atcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggc tccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcggga agttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacgga agttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcag acagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaat

SEQ ID NO:137(L27V CP 34 TEV)

MLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDG VTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAG SSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQN

SEQ ID NO:138(L27V CP 40 TEV)

Atgccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccg tatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggat gatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatc gactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggacc ctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgta accatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagt tctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagt tctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctg gaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaact

SEQ ID NO:139(L27V CP 40 TEV)

MPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMI DYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGS SGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVT

SEQ ID NO:140(L27V CP 43 TEV)

Atgaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttc ggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaac ggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaac ggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtgga gagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtgga atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtc cttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaa

SEQ ID NO:141(L27V CP 43 TEV)

MRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYF GRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGG EPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQ

SEQ ID NO:142(L27V CP 44 TEV)

Atgattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctg agcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcacttt aaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggc aacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacgga gtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagag cctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatg gtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtcctt gagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaagg

SEQ ID NO:143(L27V CP 44 TEV)

MIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG RPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGE PTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR

SEQ ID NO:144(L27V CP 45 TEV)

Atggtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagc ggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaag gtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacgg ccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaac aaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtg accggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcct actactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtg tttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgag cagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt

SEQ ID NO:145(L27V CP 45 TEV)

MVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGR PYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEP TTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI

SEQ ID NO:146(L27V CP 46 TEV)

Atgctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg attctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgacc ggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctact actgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgttt acactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcag ggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt

SEQ ID NO:147(L27V CP 46 TEV)

MLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRP YEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPT TENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI

SEQ ID NO:148(L27V CP 47 TEV)

atgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgat cagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatt ctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtat gaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggc tggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactact gagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttaca ctcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggc ggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctg

SEQ ID NO:149(L27V CP 47 TEV)

MSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPY EGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTT ENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVL

SEQ ID NO:150(L27V CP 48 TEV)

atgggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcag atgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctg cactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaa ggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatc gacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctgg cggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgag aacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactc gaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggt gtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcg

SEQ ID NO:151(L27V CP 48 TEV)

MGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYE GIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTE NLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLS

SEQ ID NO:152(L27V CP 49 TEV)

atggaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatg ggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcac tatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggc atcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgac gagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcgg ctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaac ttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaa gatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtg tccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggt

SEQ ID NO:153(L27V CP 49 TEV)

MENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEG IAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTEN LYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSG

SEQ ID NO:154(L27V CP 50 TEV)

atgaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggc cagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactat ggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatc gccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgag cgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctg tgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttg tacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagat ttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtcc agtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaa

SEQ ID NO:155(L27V CP 50 TEV)

MNGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGI AVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENL YFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGE

SEQ ID NO:156(L27V CP 51 TEV)

atgggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccag atcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggc acactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgcc gtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgc ctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgc gagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtac ttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttc gtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagt ttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaac

SEQ ID NO:157(L27V CP 51 TEV)

MGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIA VFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLY FQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGEN

SEQ ID NO:158(L27V CP 52 TEV)

atgctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatc gaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcaca ctggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtg ttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctg atcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgag cgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttc cagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgta ggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttg tttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggc

SEQ ID NO:159(L27V CP 52 TEV)

MLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAV FDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYF QSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENG

SEQ ID NO:160(L27V CP 53 TEV)

atgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaa aaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactg gtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttc gacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatc aaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgc attttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccag agcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggg gactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgttt cagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctg

SEQ ID NO:161(L27V CP 53 TEV)

MKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVF DGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQ SDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGL

SEQ ID NO:162(L27V CP 54 TEV)

atgatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggta atcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgac ggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaac cccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcatt ttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagc gataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggac tggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcag aatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaag

SEQ ID NO:163(L27V CP 54 TEV)

MIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFD GKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQS DNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLK

SEQ ID NO:164(L27V CP 55 TEV)

atggacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatt tttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatc gacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggc aaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacccc gacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttg gcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgat aacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactgg cggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaat ctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatc

SEQ ID NO:165(L27V CP 55 TEV)

MDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDG KKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSD NGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKI

SEQ ID NO:166(L27V CP 56 TEV)

Atgatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaattttt aaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgac ggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaa aagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgac ggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcg ggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataac ggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcgg cagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcga

SEQ ID NO:167(L27V CP 56 TEV)

MIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGK KITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDN GSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKID

SEQ ID NO:168(L27V CP 58 TEV)

atggtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtg gtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggtt acgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatc actgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctcc ctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagt tctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagt tctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagaca gccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtg tccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccat

SEQ ID NO:169(L27V CP 58 TEV)

MVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKI TVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGS SGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIH

SEQ ID NO:170(L27V CP 64 TEV)

atgggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgat catcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgac tatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctg tggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaacc atcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttct ggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttct ggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggac caagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaa aggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaa

SEQ ID NO:171(L27V CP 64 TEV)

MGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTL WNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSS GGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE

SEQ ID NO:172(L27V CP 67 TEV)

atgggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcacttt aaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggc aacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacgga gtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagag cctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatg gtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtcctt gagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtc ctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagc

SEQ ID NO:173(L27V CP 67 TEV)

MGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNG NKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGM VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLS

SEQ ID NO:174(L27V CP 70 TEV)

atgatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatt ctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtat gaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaatt atcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggc tggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactact gagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttaca ctcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggc ggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggt gaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcag

SEQ ID NO:175(L27V CP 70 TEV)

MMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKI IDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFT LEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQ

SEQ ID NO:176(L27V CP 73 TEV)

atgatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactat ggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatc gccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgag cgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctg tgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttg tacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagat ttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtcc agtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggc ctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccag

SEQ ID NO:177(L27V CP 73 TEV)

MIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDE RLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLED FVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQ

SEQ ID NO:178(L27V CP 76 TEV)

atgatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactg gtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttc gacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatc aaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgc attttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccag agcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggg gactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgttt cagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatc gacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaa

SEQ ID NO:179(L27V CP 76 TEV)

MIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLI NPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVG DWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK

SEQ ID NO:180(L27V CP 79 TEV)

Atggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgac ggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaa aagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgac ggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcg ggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataac ggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcgg cagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccat gtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaag

SEQ ID NO:181(L27V CP 79 TEV)

MVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPD GSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWR QTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFK

SEQ ID NO:182(L27V CP 80 TEV)

atggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggg gttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaag atcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggc tccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcggga agttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacgga agttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcag acagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggg gtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtc atcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtg

SEQ ID NO:183(L27V CP 80 TEV)

MVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDG SLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQ TAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKV

SEQ ID NO:184(L27V CP 81 TEV)

atgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggtt acgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatc actgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctcc ctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagt tctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagt tctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagaca gccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtg tccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatc atcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtg

SEQ ID NO:185(L27V CP 81 TEV)

MYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGS LLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQT AGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVV

SEQ ID NO:186(L27V CP 82 TEV)

atgcctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacg ccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcact gtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctg ctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttct ggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttct ggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagcc ggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtcc gtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatc ccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtac

SEQ ID NO:187(L27V CP 82 TEV)

MPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSL LFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTA GYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVY

SEQ ID NO:188(L27V CP 83 TEV)

atggtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccg aacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgta accgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctg ttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggt ggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggt ggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggc tacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgta actccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccg tatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccct

SEQ ID NO:189(L27V CP 83 TEV)

MVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLL FRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAG YNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYP

SEQ ID NO:190(L27V CP 84 TEV)

atggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacc gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtgga ggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtgga ggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctac aacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaact ccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtat gaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtg

SEQ ID NO:191(L27V CP 84 TEV)

MDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLF RVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGY NLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPV

SEQ ID NO:192(L27V CP 85 TEV)

atggatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatg atcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccggg accctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgc gtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggagga agttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggagga agttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaac ctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccg atccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaa ggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggat

SEQ ID NO:193(L27V CP 85 TEV)

MDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYN LDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVD

SEQ ID NO:194(L27V CP 86 TEV)

atgcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatc gactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggacc ctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgta accatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagt tctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagt tctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctg gaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgat

SEQ ID NO:195(L27V CP 86 TEV)

MHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRV TINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDD

SEQ ID NO:196(L27V CP 87 TEV)

Atgcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgac tatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctg tggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaacc atcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttct ggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttct ggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggac caagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaa aggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctg agcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcat

SEQ ID NO:197(L27V CP 87 TEV)

MHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVT INGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLD QVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDH

SEQ ID NO:198(L27V CP 88 TEV)

atgtttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactat ttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtgg aacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatc aacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggt ggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggt ggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaa gtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaagg attgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagc ggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac

SEQ ID NO:199(L27V CP 88 TEV)

MFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTI NGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQ VLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH

SEQ ID NO:200(L27V CP 91 TEV)

atgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacgg ccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaac aaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtg accggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcct actactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtg tttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgag cagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctg agcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcag atgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg

SEQ ID NO:201(L27V CP 91 TEV)

MILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGV TGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLE QGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKV

SEQ ID NO:202(L27V CP 94 TEV)

atgtatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaa ggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatc gacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctgg cggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgag aacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactc gaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggt gtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaa aacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccag atcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcacg

SEQ ID NO:203(L27V CP 94 TEV)

MYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW RLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGG VSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILH

SEQ ID NO:204(L27V CP 95 TEV)

atgggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggc atcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgac gagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcgg ctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaac ttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaa gatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtg tccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaac ggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatc gaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactat

SEQ ID NO:205(L27V CP 95 TEV)

MGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR LCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGV SSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHY

SEQ ID NO:206(L27V CP 97 TEV)

atgctggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgcc gtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgc ctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgc gagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtac ttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttc gtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagt ttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctg aagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcaca

SEQ ID NO:207(L27V CP 97 TEV)

MLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLC ERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSS LFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGT

SEQ ID NO:208(L27V CP 100 TEV)

Atggacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgac ggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaac cccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcatt ttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagc gataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggac tggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcag aatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgac atccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaag gtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatc

SEQ ID NO:209(L27V CP 100 TEV)

MDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERI LAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQ NLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVI

SEQ ID NO:210(L27V CP 101 TEV)

atgggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggc aaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacccc gacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttg gcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgat aacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactgg cggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaat ctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatc catgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtg gtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgac

SEQ ID NO:211(L27V CP 101 TEV)

MGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERIL AGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQN LGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVID

SEQ ID NO:212(L27V CP 102 TEV)

atggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaa aagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgac ggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcg ggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataac ggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcgg cagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccat gtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtg taccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggg

SEQ ID NO:213(L27V CP 102 TEV)

MVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA GSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDG

SEQ ID NO:214(L27V CP 103 TEV)

atgacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaag atcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggc tccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcggga agttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacgga agttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcag acagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggg gtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtc atcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtac cctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggtt

SEQ ID NO:215(L27V CP 103 TEV)

MTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAG SSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLG VSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV

SEQ ID NO:216(L27V CP 104 TEV)

atgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatc actgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctcc ctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagt tctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagt tctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagaca gccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtg tccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatc atcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccct gtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacg

SEQ ID NO:217(L27V CP 104 TEV)

MPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGS SGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGV SVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT

SEQ ID NO:218(L27V CP 105 TEV)

atgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcact gtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctg ctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttct ggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttct ggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagcc ggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtcc gtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatc ccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtg gatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacg

SEQ ID NO:219(L27V CP 105 TEV)

MNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSS GGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVS VTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVT

SEQ ID NO:220(L27V CP 106 TEV)

atgatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgta accgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctg ttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggt ggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggt ggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggc tacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgta actccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccg tatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggat gatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaac

SEQ ID NO:221(L27V CP 106 TEV)

MMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSG GGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSV TPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPN

SEQ ID NO:222(L27V CP 109 TEV)

atgtatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggacc ctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgta accatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagt tctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagt tctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctg gaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgac

SEQ ID NO:223(L27V CP 109 TEV)

MYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGS SGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPI QRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMID

SEQ ID NO:224(L27V CP 112 TEV)

atgcggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaac ggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaac ggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtgga gagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtgga atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtc cttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg attctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga

SEQ ID NO:225(L27V CP 112 TEV)

MRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGG EPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI VLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFG

SEQ ID NO:226(L27V CP 115 TEV)

atggaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgacc ggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctact actgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgttt acactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcag ggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagc ggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatg ggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcac tatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtat

SEQ ID NO:227(L27V CP 115 TEV)

MEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPT TENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLS GENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPY

SEQ ID NO:228(L27V CP 120 TEV)

atgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgc ctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgc gagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtac ttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttc gtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagt ttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctg aagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggta atcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgtggcc

SEQ ID NO:229(L27V CP 120 TEV)

MFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLY FQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGL KIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIVA

SEQ ID NO:230(L27V CP 121 TEV)

atggacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctg atcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgag cgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttc cagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgta ggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttg tttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaag atcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatt tttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatc gacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttc

SEQ ID NO:231(L27V CP 121 TEV)

MDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYF QSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLK IDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVF

SEQ ID NO:232(L27V CP 123 TEV)

atgaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaac cccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcatt ttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagc gataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggac tggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcag aatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgac atccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaag gtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggg gttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggc

SEQ ID NO:233(L27V CP 123 TEV)

MKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQS DNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKID IHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDG

SEQ ID NO:234(L27V CP 124 TEV)

Atgaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacccc gacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttg gcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgat aacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactgg cggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaat ctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatc catgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtg gtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggtt acgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaa

SEQ ID NO:235(L27V CP 124 TEV)

MKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSD NGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDI HVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGK

SEQ ID NO:236(L27V CP 125 TEV)

atgatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgac ggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcg ggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataac ggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcgg cagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccat gtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtg taccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacg ccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaag

SEQ ID NO:237(L27V CP 125 TEV)

MITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDN GSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIH VIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKK

SEQ ID NO:238(L27V CP 129 TEV)

atggggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctg ttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggt ggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggt ggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggc tacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgta actccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccg tatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggat gatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatc gactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacc

SEQ ID NO:239(L27V CP 129 TEV)

MGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSG GGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIP YEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVT

SEQ ID NO:240(L27V CP 130 TEV)

atgaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtgga ggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtgga ggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctac aacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaact ccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtat gaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgat catcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgac tatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccggg

SEQ ID NO:241(L27V CP 130 TEV)

MTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGG GSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPY EGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTG

SEQ ID NO:242(L27V CP 131 TEV)

atgctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgc gtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggagga agttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggagga agttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaac ctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccg atccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaa ggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcat cactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactat ttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggacc

SEQ ID NO:243(L27V CP 131 TEV)

MLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGG SSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYE GLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGT

SEQ ID NO:244(L27V CP 133 TEV)

atgaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaacc atcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttct ggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttct ggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggac caagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaa aggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctg agcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcacttt aaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggg

SEQ ID NO:245(L27V CP 133 TEV)

MNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSS GGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGL SGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLW

SEQ ID NO:246(L27V CP 136 TEV)

atgaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacgga gtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagag cctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatg gtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtcctt gagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtc ctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgat cagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatt ctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtat gaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaac

SEQ ID NO:247(L27V CP 136 TEV)

MKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGM VFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGD QMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGN

SEQ ID NO:248(L27V CP 139 TEV)

atggacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggc tggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactact gagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttaca ctcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggc ggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggt gaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggc cagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactat ggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatc gccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgag cgc

SEQ ID NO:249(L27V CP 139 TEV)

MDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFT LEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMG QIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDE R

SEQ ID NO:250(L27V CP 140 TEV)

atggagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctgg cggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgag aacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactc gaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggt gtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaa aacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccag atcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggc acactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgcc gtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgac

SEQ ID NO:251(L27V CP 140 TEV)

MERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTL EDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQ IEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIID

SEQ ID NO:252(L27V CP 141 TEV)

atgcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcgg ctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaac ttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaa gatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtg tccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaac ggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatc gaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcaca ctggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtg ttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgag

SEQ ID NO:253(L27V CP 141 TEV)

MRLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLE DFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQI EKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDE

SEQ ID NO:254(L27V CP 142 TEV)

atgctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctg tgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttg tacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagat ttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtcc agtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggc ctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaa aaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactg gtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttc gacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgc

SEQ ID NO:255(L27V CP 142 TEV)

MLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLED FVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIE KIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDER

SEQ ID NO:256(L27V CP 143 TEV)

atgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgc gagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtac ttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttc gtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagt ttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctg aagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggta atcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgac ggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctg

SEQ ID NO:257(L27V CP 143 TEV)

MINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDF VGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEK IFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERL

SEQ ID NO:258(L27V CP 144 TEV)

atgaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgag cgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttc cagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgta ggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttg tttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaag atcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatt tttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatc gacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggc aaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatc

SEQ ID NO:259(L27V CP 144 TEV)

MNPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFV GDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKI FKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLI

SEQ ID NO:260(L27V CP 145 TEV)

atgcccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgc attttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccag agcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggg gactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgttt cagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatc gacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaattttt aaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgac ggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaa aagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaac

SEQ ID NO:261(L27V CP 145 TEV)

MPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVG DWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIF KVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLIN

SEQ ID NO:262(L27V CP 146 TEV)

atggacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcatt ttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagc gataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggac tggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcag aatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgac atccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaag gtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggg gttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaag atcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacccc

SEQ ID NO:263(L27V CP 146 TEV)

MDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGD WRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFK VVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINP

SEQ ID NO:264(L27V CP 147 TEV)

atgggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttg gcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgat aacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactgg cggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaat ctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatc catgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtg gtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggtt acgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatc actgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgac

SEQ ID NO:265(L27V CP 147 TEV)

MGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDW RQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKV VYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPD

SEQ ID NO:266(L27V CP 148 TEV)

atgtccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcg ggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataac ggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcgg cagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccat gtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtg taccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacg ccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcact gtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggc

SEQ ID NO:267(L27V CP 148 TEV)

MSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWR QTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVV YPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDG

SEQ ID NO:268(L27V CP 149 TEV)

atgctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcggga agttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacgga agttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcag acagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggg gtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtc atcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtac cctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccg aacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgta accgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctcc

SEQ ID NO:269(L27V CP 149 TEV)

MLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQ TAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVY PVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGS

SEQ ID NO:270(L27V CP 150 TEV)

atgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagt tctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagt tctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagaca gccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtg tccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatc atcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccct gtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacc gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctg

SEQ ID NO:271(L27V CP 150 TEV)

MLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQT AGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYP VDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSL

SEQ ID NO:272(L27V CP 151 TEV)

atgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttct ggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttct ggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagcc ggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtcc gtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatc ccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtg gatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatg atcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccggg accctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctg

SEQ ID NO:273(L27V CP 151 TEV)

MFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTA GYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPV DDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLL

SEQ ID NO:274(L27V CP 154 TEV)

atgaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggagga agttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggagga agttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaac ctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccg atccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaa ggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcat cactttaaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactat ttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtgg aacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgta

SEQ ID NO:275(L27V CP 154 TEV)

MTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYN LDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDH HFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRV

SEQ ID NO:276(L27V CP 156 TEV)

atgaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttct ggtggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttct ggtggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggac caagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaa aggattgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctg agcggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcacttt aaggtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggc aacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatc

SEQ ID NO:277(L27V CP 156 TEV)

MNGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLD QVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHF KVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTI

SEQ ID NO:278(L27V CP 157 TEV)

atgggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggt ggagagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggt ggaatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaa gtccttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaagg attgtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagc ggcgatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaag gtgattctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacgg ccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaac aaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaac

SEQ ID NO:279(L27V CP 157 TEV)

MGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQ VLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFK VILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN

SEQ ID NO:280(L27V CP 158 TEV)

atggtgaccggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtgga gagcctactactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtgga atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtc cttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg attctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacgga

SEQ ID NO:281(L27V CP 158 TEV)

MVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQV LEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKV ILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTING

SEQ ID NO:282(L27V CP 160 TEV)

atgggctggcggctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcct actactgagaacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtg tttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgag cagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctg agcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcag atgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctg cactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaa ggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatc gacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgacc

SEQ ID NO:283(L27V CP 160 TEV)

MGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLE QGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVIL HYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVT

SEQ ID NO:284(L27V CP 163 TEV)

atgctgtgcgagcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgag aacttgtacttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactc gaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggt gtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaa aacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccag atcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggc acactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgcc gtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgc ctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcgg

SEQ ID NO:285(L27V CP 163 TEV)

MLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGG VSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYG TLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR

SEQ ID NO:286(L27V CP 166 TEV)

Atgcgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtac ttccagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttc gtaggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagt ttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctg aagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaa atttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggta atcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgac ggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaac cccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgag

SEQ ID NO:287(L27V CP 166 TEV)

MRILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSS LFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLV IDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCE

SEQ ID NO:288(pCA 9 FKBP-L27V02A 157-169)

gagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtgg tgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagcccttta agtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggtc agagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatcccac cacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtggaggca gcggtggagtgaccggctggcggctgtgcgaacgcattctggcg

SEQ ID NO:289(pCA 9 FKBP-L27V02A 157-169)

GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVG QRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGGVTGWRLCERILA

SEQ ID NO:290(pCA 10 L27V02A 1-156-FRB)

atggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgacc ggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctg gaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttg catgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagat ttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcc tgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:291(pCA 10 L27V02A 1-156-FRB)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPL HAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:292(pCA 26 FKBP-L27V02A 1-156)

ggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgcgtg gtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagcccttt aagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtgggt cagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatccca ccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtggaggc agcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggac caagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaa aggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctg agcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcacttt aaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcgga cggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggc aacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaac

SEQ ID NO:293(pCA 26 FKBP-L27V02A 1-156)

GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVG QRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNLD QVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHF KVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN

SEQ ID NO:294(pCA 25 L27V02A 157-169-FRB)

atgggagtgaccggctggcggctgtgcgaacgcattctggcgggaggaggtggctcaggtggagggagc tccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggg gaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccag actctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcagg aagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccga cgaatctca

SEQ ID NO:295(pCA 25 L27V02A 157-169-FRB)

MGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQ TLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:296(pCA 3 FKBP-L27V02A 103-169)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggc aaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacccc gacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctg gc

SEQ ID NO:297(pCA 3 FKBP-L27V02A 103-169)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGTPNMIDYFGRPYEGIAVFDG KKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERIL

SEQ ID NO:298(pCA 4 L27V02A 1-102-FRB)

atggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttggaggaggtggctcaggtggagggagctccgtg gccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaagg aacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactctg aaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtac atgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatc tca

SEQ ID NO:299(pCA 4 L27V02A 1-102-FRB)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGTPNMIDYFGRPYEGIAVFDG KKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERIL

SEQ ID NO:300(pCA 19 L27V02A 103-169-FRB)

atgacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaag atcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggc tccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcggga ggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctggaa gaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcat gctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagattta atggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctgg gacctctattatcatgtgttccgacgaatctca

SEQ ID NO:301(pCA 19 L27V02A 103-169-FRB)

MTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAG GGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDL MEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:302(pCA 20 FKBP-L27V02A 1-102)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggggtt

SEQ ID NO:303(pCA 20 FKBP-L27V02A 1-102)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVIDGV

SEQ ID NO:304(pCA 11 FKBP-L27V02A 84-169)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtgatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacg ccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcact gtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctg ctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcg

SEQ ID NO:305(pCA 11 FKBP-L27V02A 84-169)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGDDHHFKVILHYGTLVIDGVT PNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

SEQ ID NO:306(pCA 12 L27V02A 1-83-FRB)

atggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtgggaggaggtggctcaggtgga gggagctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtac tttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggc ccccagactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtgg tgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtg ttccgacgaatctca

SEQ ID NO:307(pCA 12 L27V02A 1-83-FRB)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERG PQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

308(pCA 14L 27V02A 1-83 (unfused))

atggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtg

309(pCA 14L 27V02A 1-83 (unfused))

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPV

310(pCA 13L 27V02A 84-169 (unfused))

atggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcg

311(pCA 13L 27V02A 84-169 (unfused))

MDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLF RVTINGVTGWRLCERILA

SEQ ID NO:312(pCA 28 FKBP 1-83)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtg

SEQ ID NO:313(pCA 28 FKBP 1-83)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPV

SEQ ID NO:314(pCA 27 L27V02A 84-169 FRB)

atggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgggaggaggtggctca ggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgt ttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaa cggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaa gagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattat catgtgttccgacgaatctca

SEQ ID NO:315(pCA 27 L27V02A 84-169 FRB)

MDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLF RVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMME RGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:316(HT7-Keap1)

atggcagaaatcggtactggctttccattcgacccccattatgtggaagtcctgggcgagcgcatgcac tacgtcgatgttggtccgcgcgatggcacccctgtgctgttcctgcacggtaacccgacctcctcctac gtgtggcgcaacatcatcccgcatgttgcaccgacccatcgctgcattgctccagacctgatcggtatg ggcaaatccgacaaaccagacctgggttatttcttcgacgaccacgtccgcttcatggatgccttcatc gaagccctgggtctggaagaggtcgtcctggtcattcacgactggggctccgctctgggtttccactgg gccaagcgcaatccagagcgcgtcaaaggtattgcatttatggagttcatccgccctatcccgacctgg gacgaatggccagaatttgcccgcgagaccttccaggccttccgcaccaccgacgtcggccgcaagctg atcatcgatcagaacgtttttatcgagggtacgctgccgatgggtgtcgtccgcccgctgactgaagtc gagatggaccattaccgcgagccgttcctgaatcctgttgaccgcgagccactgtggcgcttcccaaac gagctgccaatcgccggtgagccagcgaacatcgtcgcgctggtcgaagaatacatggactggctgcac cagtcccctgtcccgaagctgctgttctggggcaccccaggcgttctgatcccaccggccgaagccgct cgcctggccaaaagcctgcctaactgcaaggctgtggacatcggcccgggtctgaatctgctgcaagaa gacaacccggacctgatcggcagcgagatcgcgcgctggctgtcgacgctcgagatttccggcgagcca accactgaggatctgtactttcagagcgataacgcgatcgctttcgaaggagatagaaccatgcagcca gatcccaggcctagcggggctggggcctgctgccgattcctgcccctgcagtcacagtgccctgagggg gcaggggacgcggtgatgtacgcctccactgagtgcaaggcggaggtgacgccctcccagcatggcaac cgcaccttcagctacaccctggaggatcataccaagcaggcctttggcatcatgaacgagctgcggctc agccagcagctgtgtgacgtcacactgcaggtcaagtaccaggatgcaccggccgcccagttcatggcc cacaaggtggtgctggcctcatccagccctgttttcaaggccatgttcaccaacgggctgcgggagcag ggcatggaggtggtgtccattgagggtatccaccccaaggtcatggagcgcctcattgaattcgcctac acggcctccatctccatgggcgagaagtgtgtcctccacgtcatgaacggcgctgtcatgtaccagatc gacagcgttgtccgtgcctgcagtgacttcctggtgcagcagctggaccccagcaatgccatcggcatc gccaacttcgctgagcagattggctgtgtggagttgcaccagcgtgcccgggagtacatctacatgcat tttggggaggtggccaagcaagaggagttcttcaacctgtcccactgccaactggtgaccctcatcagc cgggacgacctgaacgtgcgctgcgagtccgaggtcttccacgcctgcatcaactgggtcaagtacgac tgcgaacagcgacggttctacgtccaggcgctgctgcgggccgtgcgctgccactcgttgacgccgaac ttcctgcagatgcagctgcagaagtgcgagatcctgcagtccgactcccgctgcaaggactacctggtc aagatcttcgaggagctcaccctgcacaagcccacgcaggtgatgccctgccgggcgcccaaggtgggc cgcctgatctacaccgcgggcggctacttccgacagtcgctcagctacctggaggcttacaaccccagt aacggcacctggctccggttggcggacctgcaggtgccgcggagcggcctggccggctgcgtggtgggc gggctgttgtacgccgtgggcggcaggaacaactcgcccgacggcaacaccgactccagcgccctggac tgttacaaccccatgaccaatcagtggtcgccctgcgcccccatgagcgtgccccgtaaccgcatcggg gtgggggtcatcgatggccacatctatgccgtcggcggctcccacggctgcatccaccacaacagtgtg gagaggtatgagccagagcgggatgagtggcacttggtggccccaatgctgacacgaaggatcggggtg ggcgtggctgtcctcaatcgtctgctttatgccgtggggggctttgacgggacaaaccgccttaattca gctgagtgttactacccagagaggaacgagtggcgaatgatcacagcaatgaacaccatccgaagcggg gcaggcgtctgcgtcctgcacaactgtatctatgctgctgggggctatgatggtcaggaccagctgaac agcgtggagcgctacgatgtggaaacagagacgtggactttcgtagcccccatgaagcaccggcgaagt gccctggggatcactgtccaccaggggagaatctacgtccttggaggctatgatggtcacacgttcctg gacagtgtggagtgttacgacccagatacagacacctggagcgaggtgacccgaatgacatcgggccgg agtggggtgggcgtggctgtcaccatggagccctgccggaagcagattgaccagcagaactgtacctgt tacgtagtt

SEQ ID NO:317(Nrf2)

TCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATT GCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTG GCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGA GTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGAC GTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTA TTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGT CAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCA GTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGG ATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCA CCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCG TGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCACTAGAAGCTTTATTGC GGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTG CAGAAGTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATA GAAACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACAT CCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTATTAA TACGACTCACTATAGGGCTAGCGATCGCCATGATGGACTTGGAGCTGCCGCCGCCGGGACTCCCGTCCC AGCAGGACATGGATTTGATTGACATACTTTGGAGGCAAGATATAGATCTTGGAGTAAGTCGAGAAGTAT TTGACTTCAGTCAGCGACGGAAAGAGTATGAGCTGGAAAAACAGAAAAAACTTGAAAAGGAAAGACAAG AACAACTCCAAAAGGAGCAAGAGAAAGCCTTTTTCGCTCAGTTACAACTAGATGAAGAGACAGGTGAAT TTCTCCCAATTCAGCCAGCCCAGCACATCCAGTCAGAAACCAGTTCTCTCGGTGGTTCAGGTGGTGGCG GGAGCGGTGGAGGGAGCAGCGGTGGAGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAG CCGGCTACAACCTGGACCAAGTCCTTGAGCAGGGCGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGT CCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAACGGCCTGAAGATCGACATCCATGTCATCA TCCCGTATGAAGGTCTGAGCGGCGATCAGATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTG TGGATGATCATCACTTTAAGGTGATTCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACA TGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACCG GGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCC GCGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAGCGCATTTTGGCGTAAGGCCGCGACTCTA GAGTCGACCTGCAGGCATGCAAGCTGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGC TGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCGGCCGCTTCGAGCAGACATGATAAGATACA TTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATG CTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTA TGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTTAAGCAAGTAAAACCTCTACAAATGTGGTAAAA TCGAATTTTAACAAAATATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCG GTATTTCACACCGCATACGCGGATCTGCGCAGCACCATGGCCTGAAATAACCTCTGAAAGAGGAACTTG GTTAGGTACCTTCTGAGGCGGAAAGAACCAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCC AGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTC CCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCC CCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAAT TTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTT TTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTAATTAACTGTTGACAATTAATCATCGGCATAGTATATC GGCATAGTATAATACGACAAGGTGAGGAACTAAACCCAGGAGGCAGATCATGATTGAACAAGATGGATT GCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGG CTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCT GTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCC TTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGG GCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCG GCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACG TACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGC CGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGC CTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGT GGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGC TGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCT TGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCA CGATGGCCGCAATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAATCGATAGC GATAAGGATCCTCTTTGCGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTGACATTCATCCGGGG TCAGCACCGTTTCTGCGGACTGGCTTTCTACCCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATC CACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAA AAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTC AAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGT GCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGC GCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGT GCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGT AAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGG TGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGC TCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGG TAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATTTCAAGAAGATCCTTT GATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATT ATCAAAAAGGATCTTCACCTAGATCCTTTTATAGTCCGGAAATACAGGAACGCACGCTGGATGGCCCTT CGCTGGGATGGTGAAACCATGAAAAATGGCAGCTTCAGTGGATTAAGTGGGGGTAATGTGGCCTGTACC CTCTGGTTGCATAGGTATTCATACGGTTAAAATTTATCAGGCGCGATTGCGGCAGTTTTTCGGGTGGTT TGTTGCCATTTTTACCTGTCTGCTGCCGTGATCGCGCTGAACGCGTTTTAGCGGTGCGTACAATTAAGG GATTATGGTAAATCCACTTACTGTCTGCCCTCGTAGCCATCGAGATAAACCGCAGTACTCCGGCCACGA TGCGTCCGGCGTAGAGGATCGAGATCT

SEQ ID NO:318(L27V02B)

ATGGTCTTCACACTCGAAGATTTCGTAGGTGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTC CTTGAACAGGGTGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATT GTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGC GACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTG ATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCG TATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACTGGGACCCTGTGGAACGGCAACAAA ATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACC GGCTGGCGGCTGTGCGAACGCATTCTGGCG

SEQ ID NO:319(L27V01)

ATGGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTACAACCTGGACCAAGTC CTTGAACAGGGCGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATT GTCCTGAGCGGTGAAAACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGC GATCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTG ATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCG TATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACCGGGACCCTGTGGAACGGCAACAAA ATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGCGTAACCATCAACGGAGTGACC GGCTGGCGGCTGTGCGAGCGCATTTTGGCG

SEQ ID NO:320(L27V01-PEST00)

ATGGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTACAACCTGGACCAAGTC CTTGAACAGGGCGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATT GTCCTGAGCGGTGAAAACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGC GATCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTG ATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCG TATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACCGGGACCCTGTGGAACGGCAACAAA ATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGCGTAACCATCAACGGAGTGACC GGCTGGCGGCTGTGCGAGCGCATTTTGGCGAATTCACACGGCTTTCCGCCCGAGGTTGAAGAGCAAGCC GCCGGTACATTGCCTATGTCCTGCGCACAAGAAAGCGGTATGGACCGGCACCCAGCCGCTTGTGCTTCA GCTCGCATCAACGTC

SEQ ID NO:321(IL601-L27V01)

ATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGCCTGCTCCTGGTGTTGCCT GCTGCCTTCCCTGCCCCAGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTAC AACCTGGACCAAGTCCTTGAACAGGGCGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACT CCGATCCAAAGGATTGTCCTGAGCGGTGAAAACGGCCTGAAGATCGACATCCATGTCATCATCCCGTAT GAAGGTCTGAGCGGCGATCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGAT CATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGAC TATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACCGGGACCCTG TGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGCGTAACC ATCAACGGAGTGACCGGCTGGCGGCTGTGCGAGCGCATTTTGGCG

SEQ ID NO:322(L27V02A)

ATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTC CTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATT GTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGC GACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTG ATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCG TATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAA ATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACC GGCTGGCGGCTGTGCGAACGCATTCTGGCG

SEQ ID NO:323(L27V02A-PEST01)

ATGGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTC CTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATT GTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGC GACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTG ATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCG TATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAA ATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACC GGCTGGCGGCTGTGCGAACGCATTCTGGCGAATTCTCACGGCTTTCCGCCTGAGGTTGAAGAGCAAGCC GCCGGTACATTGCCTATGTCCTGCGCACAAGAAAGCGGTATGGACCGGCACCCAGCCGCTTGTGCTTCA GCTCGCATCAACGTC

SEQ ID NO:324(IL601-L27V02A)

ATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGCCTGCTCCTGGTGTTGCCT GCTGCCTTCCCTGCCCCAGTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTAC AACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACT CCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTAT GAAGGTCTGAGCGGCGACCAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGAT CATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGAC TATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTG TGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACC ATCAACGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCG

SEQ ID NO:325(L27V03)

ATGGTCTTCACACTCGAAGATTTCGTAGGTGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTT CTTGAACAGGGTGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCAATCCAGAGGATA GTCCTGAGTGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCTTATGAAGGTCTGAGCGGC GATCAGATGGGGCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTG ATCCTGCACTATGGCACACTGGTAATCGACGGTGTTACGCCGAACATGATCGACTATTTCGGACGGCCG TATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATTACTGTCACTGGAACCCTGTGGAATGGGAACAAA ATTATCGACGAGCGCCTGATCAACCCCGACGGCTCACTGCTGTTCCGAGTAACCATCAATGGTGTTACC GGCTGGCGGCTCTGCGAACGCATTCTAGCA

SEQ ID NO:326(L27V03-PEST02)

ATGGTCTTCACACTCGAAGATTTCGTAGGTGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTT CTTGAACAGGGTGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCAATCCAGAGGATA GTCCTGAGTGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCTTATGAAGGTCTGAGCGGC GATCAGATGGGGCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTG ATCCTGCACTATGGCACACTGGTAATCGACGGTGTTACGCCGAACATGATCGACTATTTCGGACGGCCG TATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATTACTGTCACTGGAACCCTGTGGAATGGGAACAAA ATTATCGACGAGCGCCTGATCAACCCCGACGGCTCACTGCTGTTCCGAGTAACCATCAATGGTGTTACC GGCTGGCGGCTCTGCGAACGCATTCTAGCAAATAGTCACGGCTTTCCGCCTGAGGTTGAAGAGCAAGCC GCCGGTACATTGCCTATGTCCTGCGCACAAGAAAGCGGTATGGACCGGCACCCAGCCGCTTGTGCTTCA GCTCGCATCAACGTC

SEQ ID NO:327(IL602-L27V03)

ATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTCGCCTTCTCCCTGGGCCTGCTCCTGGTGTTGCCC GCTGCCTTTCCTGCCCCAGTCTTCACACTCGAAGATTTCGTAGGTGACTGGCGACAGACAGCCGGCTAC AACCTGGACCAAGTTCTTGAACAGGGTGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACT CCAATCCAGAGGATAGTCCTGAGTGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCTTAT GAAGGTCTGAGCGGCGATCAGATGGGGCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGAT CATCACTTTAAGGTGATCCTGCACTATGGCACACTGGTAATCGACGGTGTTACGCCGAACATGATCGAC TATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATTACTGTCACTGGAACCCTG TGGAATGGGAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCACTGCTGTTCCGAGTAACC ATCAATGGTGTTACCGGCTGG

328 (Joint) SEQ ID NO

GSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGG

329(FABP consensus)

[GSAIVK]-{FE}-[FYW]-x-[LIVMF]-x-x-{K}-x-[NHG]-[FY]-[DE]-x-[LIVMFY]- [LIVM]-{N}-{G}-[LIVMAKR]

SEQ ID NO:330(OGLUC consensus sequence)

[GSAIVK]-{FE}-[FYW]-x-[LIVMFSYQ]-x-x-{K}-x-[NHGK]-x-[DE]-x-[LIVMFY]-[ LIVMWF]-x-{G}-[LIVMAKRG]

SEQ ID NO:331(9B8 PCA1 (pF5A/Met-[9B8 opt (51-169)]-GGGGSGGGSS-FRB)

Atgggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggctatcagatgggccag atcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggc acactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgcc gtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgc ctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgc gagcgcattttggcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatg tggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgag gtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcag gcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaag gacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:332(9B8 PCA1 (pF5A/Met-[9B8 opt (51-169)]-GGGGSGGGSS-FRB)

MGLKIDIHVIIPYEGLSGYQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIA VFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEM WHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVK DLTQAWDLYYHVFRRIS

SEQ ID NO:333(9B8 PCA2 (pF5A/[9B8 opt (1-50)]-GGGGSGGGSS-FRB)

Atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtc cttgagcagggcggtctgtccagtttgtttcagaaactcggggtgtccgtaacaccgatccaaaagatt gtcctgagcggtgaaaacggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgag atgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgttt gaggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaat caggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtc aaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:334(9B8 PCA2(pF5A/[9B8 opt (1-50)]-GGGGSGGGSS-FRB)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGLSSLFQKLGVSVTPIQKIVLSGENGGGGSGGGSSVAILWHE MWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNV KDLTQAWDLYYHVFRRIS

SEQ ID NO:335(9B8 PCA 3(& pF5A/FKBP-GGGSSGGGSG-[9B8 opt(51-169)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggctatcagatg ggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcac tatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggc atcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgac gagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcgg ctgtgcgagcgcattttggcg

SEQ ID NO:336(9B8 PCA 3(& pF5A/FKBP-GGGSSGGGSG-[9B8 opt(51-169)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGGLKIDIHVIIPYEGLSGYQM GQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIID ERLINPDGSLLFRVTINGVTGWRLCERILA

SEQ ID NO:337(9B8 PCA 4 (pF5A/FKBP-GGGSSGGGSG-[9B8 opt(1-50)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctg gaccaagtccttgagcagggcggtctgtccagtttgtttcagaaactcggggtgtccgtaacaccgatc caaaagattgtcctgagcggtgaaaac

SEQ ID NO:338(9B8 PCA 4 (pF5A/FKBP-GGGSSGGGSG-[9B8 opt(1-50)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGLSSLFQKLGVSVTPIQKIVLSGEN

SEQ ID NO:339(K33N+L27V+K43R+Y68D)

Atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtc cttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaacaccgatccaaaggatt gtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg attctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgacc ggctggcggctgtgcgagcgcattttggcg

SEQ ID NO:340(K33N+L27V+K43R+Y68D)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILA

SEQ ID NO:341(K33N+L27V+T39T+K43R+S66N)

Atggtgtttacactcgaagatttcgtaggggactggcggcagacagccggctacaacctggaccaagtc cttgagcagggcggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaacggcctgaagatcgacatccatgtcatcatcccgtatgaaggtctgaacggc tatcagatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg attctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgacc ggctggcggctgtgcgagcgcattttggcg

SEQ ID NO:342(K33N+L27V+T39T+K43R+S66N)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLNG YQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILA

SEQ ID NO:343(pCA 31 pCA L27V02A 45-169 FRB)

atggtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagc ggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaag gtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacgg ccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaac aaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtg accggctggcggctgtgcgaacgcattctggcgggaggaggtggctcaggtggagggagctccgtggcc atcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaac gtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaag gaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatg aaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:344(pCA 31 pCA L27V02A 45-169 FRB)

MVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGR PYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVA ILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYM KSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:345(pCA 32 FKBP L27V02A 1-44)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggatt

SEQ ID NO:346(pCA 32 FKBP L27V02A 1-44)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRI

SEQ ID NO:347(pCA 33 pCA L27V02A 46-169 FRB)

atgctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgacc ggctggcggctgtgcgaacgcattctggcgggaggaggtggctcaggtggagggagctccgtggccatc ctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtg aaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaa acatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaa tcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:348(pCA 33 pCA L27V02A 46-169 FRB)

MLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRP YEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAI LWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMK SGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:349(pCA 34 pCA FKBP 1-45 L27V02A)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtc

SEQ ID NO:350(pCA 34 pCA FKBP 1-45 L27V02A)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIV

SEQ ID NO:351(pCA 35 pCA L27V02A 100-169 FRB)

atggacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgac ggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaac cccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcatt ctggcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaa ggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggag cccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggt cgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacc caagcctgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:352(pCA 35 pCA L27V02A 100-169 FRB)

MDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERI LAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYG RDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:353(pCA 36 FKBP L27V02A 1-99)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatc

SEQ ID NO:354(pCA 36 FKBP L27V02A 1-99)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVI

SEQ ID NO:355(pCA 37 L27V02A 101-169 FRB)

atgggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggc aaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacccc gacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctg gcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggc ctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagccc ttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcga gatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaa gcctgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:356(pCA 37 L27V02A 101-169 FRB)

MGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERIL AGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGR DLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:357(pCA 38 FKBP 1-100 L27V02A)

Atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgac

SEQ ID NO:358(pCA 38 FKBP 1-100 L27V02A)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVID

SEQ ID NO:359(pCA 39 L27V02A 102-169 FRB)

Atggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaa aagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgac ggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcg ggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctg gaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttg catgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagat ttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcc tgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:360(pCA 39 L27V02A 102-169 FRB)

MVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA GGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRD LMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:361(pCA 40 FKBP L27V02A 1-101)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggg

SEQ ID NO:362(pCA 40 FKBP L27V02A 1-101)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVIDG

SEQ ID NO:363(pCA 41 L27V02A 143-169 FRB)

atgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgc gaacgcattctggcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatg tggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgag gtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcag gcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaag gacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:364(pCA 41 L27V02A 143-169 FRB)

MINPDGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFE VLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:365(pCA 42 FKBP 1-142 L27V02A)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttc ggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaac ggcaacaaaattatcgacgagcgcctg

SEQ ID NO:366(pCA 42 FKBP 1-142 L27V02A)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERL

SEQ ID NO:367(pCA 43 L27V02A 145-169 FRB)

atgcccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgc attctggcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcat gaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctg gagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctat ggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctc acccaagcctgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:368(pCA 43 L27V02A 145-169 FRB)

MPDGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVL EPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:369(pCA 44 FKBP 1-144 L27V02A)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttc ggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaac ggcaacaaaattatcgacgagcgcctgatcaac

SEQ ID NO:370(pCA 44 FKBP 1-144 L27V02A)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLIN

SEQ ID NO:371(pCA 45 L27V02A 147-169 FRB)

atgggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctg gcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggc ctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagccc ttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcga gatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaa gcctgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:372(pCA 45 L27V02A 147-169 FRB)

MGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEP LHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:373(pCA 46 FKBP-L27V02A 1-146)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagcac tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttc ggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaac ggcaacaaaattatcgacgagcgcctgatcaaccccgac

SEQ ID NO:374(pCA 46 L27V02A-FKBP 1-146)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKHFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPD

SEQ ID NO:375(pCA 47 L27V02A 148-169 FRB)

atgtccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcg ggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctg gaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttg catgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagat ttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcc tgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:376(pCA 47 L27V02A 148-169 FRB)

MSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPL HAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:377(pCA 48 FKBP-L27V02A 1-147)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttc ggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaac ggcaacaaaattatcgacgagcgcctgatcaaccccgacggc

SEQ ID NO:378(pCA 48 FKBP-L27V02A 1-147)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDG

SEQ ID NO:379(pCA 49 L27V02A 156-169 FRB)

atgaacggagtgaccggctggcggctgtgcgaacgcattctggcgggaggaggtggctcaggtggaggg agctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtacttt ggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccc cagactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgc aggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttc cgacgaatctca

SEQ ID NO:380(pCA 49 L27V02A 156-169 FRB)

MNGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGP QTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:381(pCA 50 FKBP-L27V02A 1-155)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttc ggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaac ggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatc

SEQ ID NO:382(pCA 50 FKBP-L27V02A 1-155)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTI

SEQ ID NO:383(pCA 51 L27V02A 158-169 FRB)

atggtgaccggctggcggctgtgcgaacgcattctggcgggaggaggtggctcaggtggagggagctcc gtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaa aggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagact ctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaag tacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacga atctca

SEQ ID NO:384(pCA 51 L27V02A 158-169 FRB)

MVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQT LKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:385(pCA 52 FKBP 1-157 L27V02A)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttc ggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaac ggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaac gga

SEQ ID NO:386(pCA 52 FKBP 1-157 L27V02A)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN G

SEQ ID NO:387(pCA 53 L27V02A 166-169 FRB)

atgcgcattctggcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatg tggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgag gtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcag gcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaag gacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:388(pCA 53 L27V02A 166-169 FRB)

MRILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQ AYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:389(pCA 54 FKBP L27V02A 1-165)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttc ggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaac ggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaac ggagtgaccggctggcggctgtgcgaa

SEQ ID NO:390(pCA 54 FKBP L27V02A 1-165)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN GVTGWRLCE

SEQ ID NO:391(pCA 55 FKBP L27V02A 1-47)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagc

SEQ ID NO:392(pCA 55 FKBP L27V02A 1-47)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLS

SEQ ID NO:393(pCA 56 L27V02A 48-169-FRB)

Atgggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaa atgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctg cactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaa ggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatc gacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctgg cggctgtgcgaacgcattctggcgggaggaggtggctcaggtggagggagctccgtggccatcctctgg catgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggc atgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcc tttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcaggg aatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:394(pCA 56 L27V02A 48-169-FRB)

MGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYE GIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILW HEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSG NVKDLTQAWDLYYHVFRRIS

SEQ ID NO:395(pCA 57 FKBP L27V02A 1-49)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaa

SEQ ID NO:396(pCA 57 FKBP L27V02A 1-49)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGE

SEQ ID NO:397(pCA 58 pCA L27V02A 50-169 FRB)

atgaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggc cagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactat ggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatc gccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgag cgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctg tgcgaacgcattctggcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgag atgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgttt gaggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaat caggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtc aaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:398(pCA 58 pCA L27V02A 50-169 FRB)

MNGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGI AVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHE MWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNV KDLTQAWDLYYHVFRRIS

SEQ ID NO:399(pCA 59 FKBP L27V02A 1-82)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccct

SEQ ID NO:400(pCA 59 FKBP L27V02A 1-82)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYP

SEQ ID NO:401(pCA 60 L27V02A 83-169-FRB)

atggtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccg aacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgta acagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctg ttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgggaggaggtggc tcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatct cgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatg gaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcc caagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctat tatcatgtgttccgacgaatctca

SEQ ID NO:402(pCA 60 L27V02A 83-16-FRB)

MVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLL FRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMM ERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:403(pCA 61 FKBP L27V02A 1-84)

Atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggat

SEQ ID NO:404(pCA 61 FKBP L27V02A 1-84)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVD

SEQ ID NO:405(pCA 62 L27V02A 85-169-FRB)

atggatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatg atcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacaggg accctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccga gtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctggcgggaggaggtggctcaggt ggagggagctccgtggccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttg tactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacgg ggcccccagactctgaaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagag tggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcat gtgttccgacgaatctca

SEQ ID NO:406(pCA 62 L27V02A 85-169-FRB)

MDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFR VTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMER GPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:407(pCA 63 FKBP L27V02A 1-122)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttc ggacggccgtatgaaggcatcgccgtgttcgacggc

SEQ ID NO:408(pCA 63 FKBP L27V02A 1-122)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDG

SEQ ID NO:409(pCA 64 L27V02A 123-169-FRB)

atgaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaac cccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcatt ctggcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaa ggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggag cccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggt cgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacc caagcctgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:410(pCA 64 L27V02A 123-169-FRB)

MKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHE GLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLT QAWDLYYHVFRRIS

SEQ ID NO:411(pCA 65 FKBP L27V02A 1-123)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttc ggacggccgtatgaaggcatcgccgtgttcgacggcaaa

SEQ ID NO:412(pCA 65 FKBP L27V02A 1-123)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGK

SEQ ID NO:413(pCA 66 L27V02A 124-169 FRB)

atgaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacccc gacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtgcgaacgcattctg gcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaaggc ctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggagccc ttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggtcga gatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacccaa gcctgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:414(pCA 66 L27V02A 124-169 FRB)

MKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGGGGSGGGSSVAILWHEMWHEG LEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQ AWDLYYHVFRRIS

SEQ ID NO:415(pCA 67 L27V02A 1-168)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttc ggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaac ggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaac ggagtgaccggctggcggctgtgcgaacgcattctg

SEQ ID NO:416(pCA 67 L27V02A 1-168)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttc ggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaac ggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaac ggagtgaccggctggcggctgtgcgaacgcattctg

SEQ ID NO:417(pCA 67 L27V02A 1-168)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN GVTGWRLCERIL

SEQ ID NO:418(*pCA 68 L27V02A 169 FRB)

Atggcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtggcatgaa ggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtgctggag cccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcctatggt cgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggacctcacc caagcctgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:419(*pCA 68 L27V02A 169 FRB)

MAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYG RDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:420(pCA 69 FKBP L27V02A 1-166)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttc ggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaac ggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaac ggagtgaccggctggcggctgtgcgaacgc

SEQ ID NO:421(pCA 69 FKBP L27V02A 1-166)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN GVTGWRLCER

SEQ ID NO:422(*pCA 70 L27V02A 167-169 FRB)

Atgattctggcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgagatgtgg catgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgtttgaggtg ctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaatcaggcc tatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtcaaggac ctcacccaagcctgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:423(*pCA 70 L27V02A 167-169 FRB)

MILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQA YGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:424(pCA 71 FKBP L27V02A 1-164)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttc ggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaac ggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaac ggagtgaccggctggcggctgtgc

SEQ ID NO:425(pCA 71 FKBP L27V02A 1-164)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN GVTGWRLC

SEQ ID NO:426(pCA 72 L27V02A 165-169 FRB)

atggaacgcattctggcgggaggaggtggctcaggtggagggagctccgtggccatcctctggcatgag atgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggcatgttt gaggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcctttaat caggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcagggaatgtc aaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca

427 (Pattern sequence)

GSAIVK

SEQ ID NO:428(pCA 72 L27V02A 165-169 FRB)

MERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFN QAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:429(pCA 73 FKBP L27V02A 1-162)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttc ggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaac ggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaac ggagtgaccggctggcgg

SEQ ID NO:430(pCA 73 FKBP L27V02A 1-162)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN GVTGWR

SEQ ID NO:431(pCA 74 L27V02A 163-169 FRB)

atgctgtgcgaacgcattctggcgggaggaggtggctcaggtggagggagctccgtggccatcctctgg catgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtgaaaggc atgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaaacatcc tttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaatcaggg aatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:432(pCA 74 L27V02A 163-169 FRB)

MLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETS FNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:433(pCA 75 FKBP L27V02A 1-160)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttc ggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaac ggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaac ggagtgaccggc

SEQ ID NO:434(pCA 75 FKBP L27V02A 1-160)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN GVTG

SEQ ID NO:435(pCA 76 L27V02A 161-169 FRB)

atgtggcggctgtgcgaacgcattctggcgggaggaggtggctcaggtggagggagctccgtggccatc ctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaaggaacgtg aaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactctgaaggaa acatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtacatgaaa tcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatctca

SEQ ID NO:436(pCA 76 L27V02A 161-169 FRB)

MWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKE TSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:437(pCA 77 FKBP L27V02A 1-158)

atgggagtgcaggtggaaaccatctccccaggagacgggcgcaccttccccaagcgcggccagacctgc gtggtgcactacaccgggatgcttgaagatggaaagaaatttgattcctcccgggacagaaacaagccc tttaagtttatgctaggcaagcaggaggtgatccgaggctgggaagaaggggttgcccagatgagtgtg ggtcagagagccaaactgactatatctccagattatgcctatggtgccactgggcacccaggcatcatc ccaccacatgccactctcgtcttcgatgtggagcttctaaaactggaaggaggagggagctccggtgga ggcagcggtatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttc ggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaac ggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaac ggagtg

SEQ ID NO:438(pCA 77 FKBP L27V02A 1-158)

MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSV GQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGSSGGGSGMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHH FKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN GV

SEQ ID NO:439(pCA 78 L27V02A 159-169 FRB)

Atgaccggctggcggctgtgcgaacgcattctggcgggaggaggtggctcaggtggagggagctccgtg gccatcctctggcatgagatgtggcatgaaggcctggaagaggcatctcgtttgtactttggggaaagg aacgtgaaaggcatgtttgaggtgctggagcccttgcatgctatgatggaacggggcccccagactctg aaggaaacatcctttaatcaggcctatggtcgagatttaatggaggcccaagagtggtgcaggaagtac atgaaatcagggaatgtcaaggacctcacccaagcctgggacctctattatcatgtgttccgacgaatc tca

SEQ ID NO:440(pCA 78 L27V02A 159-169 FRB)

MTGWRLCERILAGGGGSGGGSSVAILWHEMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTL KETSFNQAYGRDLMEAQEWCRKYMKSGNVKDLTQAWDLYYHVFRRIS

SEQ ID NO:441 RIIbB (nucleotide sequence)

ATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTA GTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGAT TCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAA GAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACT AACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCA TTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTA GTTGCCCTGTATGGAACGAACATGGATATTGTA

SEQ ID NO 442 RIIbB (amino acid sequence)

MYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVE ENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQL VALYGTNMDIV

SEQ ID NO:443(L27V CP 13 TEV)

atgacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatccat gtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaaggtggtg taccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggggttacg ccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcact gtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctg ctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcgggaagttct ggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacggaagttct ggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcag

SEQ ID NO:444(L27V CP 13 TEV)

MTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVV YPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSL LFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQ

SEQ ID NO:445(L27V CP 57 TEV)

Atgcatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatttttaag gtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactggtaatcgacggg gttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaag atcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggc tccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgagcgcattttggcggga agttctggtggaggaagttctggtggagagcctactactgagaacttgtacttccagagcgataacgga agttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgtaggggactggcggcag acagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttgtttcagaatctcggg gtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaagatcgacatc

SEQ ID NO:446(L27V CP 57 TEV)

MHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKK ITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGSSGGEPTTENLYFQSDNG SSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDI

SEQ ID NO:447(L27V CP 98 TEV)

atggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtg ttcgacggcaaaaagatcactgtaaccgggaccctgtggaacggcaacaaaattatcgacgagcgcctg atcaaccccgacggctccctgctgttccgcgtaaccatcaacggagtgaccggctggcggctgtgcgag cgcattttggcgggaagttctggtggaggaagttctggtggagagcctactactgagaacttgtacttc cagagcgataacggaagttctggtggaggaagttctggtggaatggtgtttacactcgaagatttcgta ggggactggcggcagacagccggctacaacctggaccaagtccttgagcagggcggtgtgtccagtttg tttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaacggcctgaag atcgacatccatgtcatcatcccgtatgaaggtctgagcggcgatcagatgggccagatcgaaaaaatt tttaaggtggtgtaccctgtggatgatcatcactttaaggtgattctgcactatggcacactg

SEQ ID NO:448(L27V CP 98 TEV)

MVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCE RILAGSSGGGSSGGEPTTENLYFQSDNGSSGGGSSGGMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSL FQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTL

449 (human protein kinase C alpha (PKCa) (nucleotide sequence)

ATGGCTGACGTTTTCCCGGGCAACGACTCCACGGCGTCTCAGGACGTGGCCAACCGCTTCGCCCGCAAA GGGGCGCTGAGGCAGAAGAACGTGCACGAGGTGAAGGACCACAAATTCATCGCGCGCTTCTTCAAGCAG CCCACCTTCTGCAGCCACTGCACCGACTTCATCTGGGGGTTTGGGAAACAAGGCTTCCAGTGCCAAGTT TGCTGTTTTGTGGTCCACAAGAGGTGCCATGAATTTGTTACTTTTTCTTGTCCGGGTGCGGATAAGGGA CCCGACACTGATGACCCCAGGAGCAAGCACAAGTTCAAAATCCACACTTACGGAAGCCCCACCTTCTGC GATCACTGTGGGTCACTGCTCTATGGACTTATCCATCAAGGGATGAAATGTGACACCTGCGATATGAAC GTTCACAAGCAATGCGTCATCAATGTCCCCAGCCTCTGCGGAATGGATCACACTGAGAAGAGGGGGCGG ATTTACCTAAAGGCTGAGGTTGCTGATGAAAAGCTCCATGTCACAGTACGAGATGCAAAAAATCTAATC CCTATGGATCCAAACGGGCTTTCAGATCCTTATGTGAAGCTGAAACTTATTCCTGATCCCAAGAATGAA AGCAAGCAAAAAACCAAAACCATCCGCTCCACACTAAATCCGCAGTGGAATGAGTCCTTTACATTCAAA TTGAAACCTTCAGACAAAGACCGACGACTGTCTGTAGAAATCTGGGACTGGGATCGAACAACAAGGAAT GACTTCATGGGATCCCTTTCCTTTGGAGTTTCGGAGCTGATGAAGATGCCGGCCAGTGGATGGTACAAG TTgCTTAACCAAGAAGAAGGTGAGTACTACAACGTACCCATTCCGGAAGGGGACGAGGAAGGAAACATG GAACTCAGGCAGAAATTCGAGAAAGCCAAACTTGGCCCTGCTGGCAACAAAGTCATCAGTCCCTCTGAA GACAGGAAACAACCTTCCAACAACCTTGACCGAGTGAAACTCACGGACTTCAATTTCCTCATGGTGTTG GGAAAGGGGAGTTTTGGAAAGGTGATGCTTGCCGACAGGAAGGGCACAGAAGAACTGTATGCAATCAAA ATCCTGAAGAAGGATGTGGTGATTCAGGATGATGACGTGGAGTGCACCATGGTAGAAAAGCGAGTCTTG GCCCTGCTTGACAAACCCCCGTTCTTGACGCAGCTGCACTCCTGCTTCCAGACAGTGGATCGGCTGTAC TTCGTCATGGAATATGTCAACGGTGGGGACCTCATGTACCACATTCAGCAAGTAGGAAAATTTAAGGAA CCACAAGCAGTATTCTATGCGGCAGAGATTTCCATCGGATTGTTCTTTCTTCATAAAAGAGGAATCATT TATAGGGATCTGAAGTTAGATAACGTCATGTTGGATTCAGAAGGACATATCAAAATTGCTGACTTTGGG ATGTGCAAGGAACACATGATGGATGGAGTCACGACCAGGACCTTCTGTGGGACTCCAGATTATATCGCC CCAGAGATAATCGCTTATCAGCCGTATGGAAAATCTGTGGACTGGTGGGCCTATGGCGTCCTGTTGTAT GAAATGCTTGCCGGGCAGCCTCCATTTGATGGTGAAGATGAAGACGAGCTATTTCAGTCTATCATGGAG CACAACGTTTCCTATCCAAAATCCTTGTCCAAGGAGGCTGTTTCTATCTGCAAAGGACTGATGACCAAA CACCCAGCCAAGCGGCTGGGCTGTGGGCCTGAGGGGGAGAGGGACGTGAGAGAGCATGCCTTCTTCCGG AGGATCGACTGGGAAAAACTGGAGAACAGGGAGATCCAGCCACCATTCAAGCCCAAAGTGTGTGGCAAA GGAGCAGAGAACTTTGACAAGTTCTTCACACGAGGACAGCCCGTCTTAACACCACCTGATCAGCTGGTT ATTGCTAACATAGACCAGTCTGATTTTGAAGGGTTCTCGTATGTCAACCCCCAGTTTGTGCACCCCATC TTACAGAGTGCAGTAGTT

450 (human protein kinase C alpha (PKCa) (amino acid sequence)

MADVFPGNDSTASQDVANRFARKGALRQKNVHEVKDHKFIARFFKQPTFCSHCTDFIWGFGKQGFQCQV CCFVVHKRCHEFVTFSCPGADKGPDTDDPRSKHKFKIHTYGSPTFCDHCGSLLYGLIHQGMKCDTCDMN VHKQCVINVPSLCGMDHTEKRGRIYLKAEVADEKLHVTVRDAKNLIPMDPNGLSDPYVKLKLIPDPKNE SKQKTKTIRSTLNPQWNESFTFKLKPSDKDRRLSVEIWDWDRTTRNDFMGSLSFGVSELMKMPASGWYK LLNQEEGEYYNVPIPEGDEEGNMELRQKFEKAKLGPAGNKVISPSEDRKQPSNNLDRVKLTDFNFLMVL GKGSFGKVMLADRKGTEELYAIKILKKDVVIQDDDVECTMVEKRVLALLDKPPFLTQLHSCFQTVDRLY FVMEYVNGGDLMYHIQQVGKFKEPQAVFYAAEISIGLFFLHKRGIIYRDLKLDNVMLDSEGHIKIADFG MCKEHMMDGVTTRTFCGTPDYIAPEIIAYQPYGKSVDWWAYGVLLYEMLAGQPPFDGEDEDELFQSIME HNVSYPKSLSKEAVSICKGLMTKHPAKRLGCGPEGERDVREHAFFRRIDWEKLENREIQPPFKPKVCGK GAENFDKFFTRGQPVLTPPDQLVIANIDQSDFEGFSYVNPQFVHPILQSAVV

451 (human Glucocorticoid Receptor (GR) (nucleotide sequence)

ATGGACTCCAAAGAATCATTAACTCCTGGTAGAGAAGAAAACCCCAGCAGTGTGCTTGCTCAGGAGAGG GGAGATGTGATGGACTTCTATAAAACCCTAAGAGGAGGAGCTACTGTGAAGGTTTCTGCGTCTTCACCC TCACTGGCTGTCGCTTCTCAATCAGACTCCAAGCAGCGAAGACTTTTGGTTGATTTTCCAAAAGGCTCA GTAAGCAATGCGCAGCAGCCAGATCTGTCCAAAGCAGTTTCACTCTCAATGGGACTGTATATGGGAGAG ACAGAAACAAAAGTGATGGGAAATGACCTGGGATTCCCACAGCAGGGCCAAATCAGCCTTTCCTCGGGG GAAACAGACTTAAAGCTTTTGGAAGAAAGCATTGCAAACCTCAATAGGTCGACCAGTGTTCCAGAGAAC CCCAAGAGTTCAGCATCCACTGCTGTGTCTGCTGCCCCCACAGAGAAGGAGTTTCCAAAAACTCACTCT GATGTATCTTCAGAACAGCAACATTTGAAGGGCCAGACTGGCACCAACGGTGGCAATGTGAAATTGTAT ACCACAGACCAAAGCACCTTTGACATTTTGCAGGATTTGGAGTTTTCTTCTGGGTCCCCAGGTAAAGAG ACGAATGAGAGTCCTTGGAGATCAGACCTGTTGATAGATGAAAACTGTTTGCTTTCTCCTCTGGCGGGA GAAGACGATTCATTCCTTTTGGAAGGAAACTCGAATGAGGACTGCAAGCCTCTCATTTTACCGGACACT AAACCCAAAATTAAGGATAATGGAGATCTGGTTTTGTCAAGCCCCAGTAATGTAACACTGCCCCAAGTG AAAACAGAAAAAGAAGATTTCATCGAACTCTGCACCCCTGGGGTAATTAAGCAAGAGAAACTGGGCACA GTTTACTGTCAGGCAAGCTTTCCTGGAGCAAATATAATTGGTAATAAAATGTCTGCCATTTCTGTTCAT GGTGTGAGTACCTCTGGAGGACAGATGTACCACTATGACATGAATACAGCATCCCTTTCTCAACAGCAG GATCAGAAGCCTATTTTTAATGTCATTCCACCAATTCCCGTTGGTTCCGAAAATTGGAATAGGTGCCAA GGATCTGGAGATGACAACTTGACTTCTCTGGGGACTCTGAACTTCCCTGGTCGAACAGTTTTTTCTAAT GGCTATTCAAGCCCCAGCATGAGACCAGATGTAAGCTCTCCTCCATCCAGCTCCTCAACAGCAACAACA GGACCACCTCCCAAACTCTGCCTGGTGTGCTCTGATGAAGCTTCAGGATGTCATTATGGAGTCTTAACT TGTGGAAGCTGTAAAGTTTTCTTCAAAAGAGCAGTGGAAGGACAGCACAATTACCTATGTGCTGGAAGG AATGATTGCATCATCGATAAAATTCGAAGAAAAAACTGCCCAGCATGCCGCTATCGAAAATGTCTTCAG GCTGGAATGAACCTGGAAGCTCGAAAAACAAAGAAAAAAATAAAAGGAATTCAGCAGGCCACTACAGGA GTCTCACAAGAAACCTCTGAAAATCCTGGTAACAAAACAATAGTTCCTGCAACGTTACCACAACTCACC CCTACCCTGGTGTCACTGTTGGAGGTTATTGAACCTGAAGTGTTATATGCAGGATATGATAGCTCTGTT CCAGACTCAACTTGGAGGATCATGACTACGCTCAACATGTTAGGAGGGCGGCAAGTGATTGCAGCAGTG AAATGGGCAAAGGCAATACCAGGTTTCAGGAACTTACACCTGGATGACCAAATGACCCTACTGCAGTAC TCCTGGATGTTTCTTATGGCATTTGCTCTGGGGTGGAGATCATATAGACAATCAAGTGCAAACCTGCTG TGTTTTGCTCCTGATCTGATTATTAATGAGCAGAGAATGACTCTACCCTGCATGTACGACCAATGTAAA CACATGCTGTATGTTTCCTCTGAGTTACACAGGCTTCAGGTATCTTATGAAGAGTATCTCTGTATGAAA ACCTTACTGCTTCTCTCTTCAGTTCCTAAGGACGGTCTGAAGAGCCAAGAGCTATTTGATGAAATTAGA ATGACCTACATCAAAGAGCTAGGAAAAGCCATTGTCAAGAGGGAAGGAAACTCCAGCCAGAACTGGCAG CGGTTTTATCAACTGACAAAACTCTTGGATTCTATGCATGAAGTGGTTGAAAATCTCCTTAACTATTGC TTCCAAACATTTTTGGATAAGACCATGAGTATTGAATTCCCCGAGATGTTAGCTGAAATCATCACCAAT CAGATACCAAAATATTCAAATGGAAATATCAAAAAACTTCTGTTTCATCAAAAGGTT

452 (human Glucocorticoid Receptor (GR) (amino acid sequence)

MDSKESLTPGREENPSSVLAQERGDVMDFYKTLRGGATVKVSASSPSLAVASQSDSKQRRLLVDFPKGS VSNAQQPDLSKAVSLSMGLYMGETETKVMGNDLGFPQQGQISLSSGETDLKLLEESIANLNRSTSVPEN PKSSASTAVSAAPTEKEFPKTHSDVSSEQQHLKGQTGTNGGNVKLYTTDQSTFDILQDLEFSSGSPGKE TNESPWRSDLLIDENCLLSPLAGEDDSFLLEGNSNEDCKPLILPDTKPKIKDNGDLVLSSPSNVTLPQV KTEKEDFIELCTPGVIKQEKLGTVYCQASFPGANIIGNKMSAISVHGVSTSGGQMYHYDMNTASLSQQQ DQKPIFNVIPPIPVGSENWNRCQGSGDDNLTSLGTLNFPGRTVFSNGYSSPSMRPDVSSPPSSSSTATT GPPPKLCLVCSDEASGCHYGVLTCGSCKVFFKRAVEGQHNYLCAGRNDCIIDKIRRKNCPACRYRKCLQ AGMNLEARKTKKKIKGIQQATTGVSQETSENPGNKTIVPATLPQLTPTLVSLLEVIEPEVLYAGYDSSV PDSTWRIMTTLNMLGGRQVIAAVKWAKAIPGFRNLHLDDQMTLLQYSWMFLMAFALGWRSYRQSSANLL CFAPDLIINEQRMTLPCMYDQCKHMLYVSSELHRLQVSYEEYLCMKTLLLLSSVPKDGLKSQELFDEIR MTYIKELGKAIVKREGNSSQNWQRFYQLTKLLDSMHEVVENLLNYCFQTFLDKTMSIEFPEMLAEIITN QIPKYSNGNIKKLLFHQKV

453(L27V02-GR/L27V02-PKCa linker (nucleotide sequence)

GGTGGTTCAGGTGGTGGCGGGAGCGGTGGAGGGAGCAGCGGTGGAGCGATCGCC

454(L27V02-GR/L27V02-PKCa linker (amino acid sequence)

GGSGGGGSGGGSSGGAIA

455(GR-L27V02/PKCa-L27V02 linker (nucleotide sequence)

TCTCTCGGTGGTTCAGGTGGTGGCGGGAGCGGTGGAGGGAGCAGCGGTGGA

456(GR-L27V02/PKCa-L27V02 linker (amino acid sequence)

SLGGSGGGGSGGGSSGG

457(GSSG linker (nucleotide sequence)

GCTCGAGCGGA

458(GSSG linker (amino acid sequence)

GSSG

459(AT1R (nucleotide sequence)

ATGATTCTCAACTCTTCTACTGAAGATGGTATTAAAAGAATCCAAGATGATTGTCCCAAAGCTGGAAGG CATAATTACATATTTGTCATGATTCCTACTTTATACAGTATCATCTTTGTGGTGGGAATATTTGGAAAC AGCTTGGTGGTGATAGTCATTTACTTTTATATGAAGCTGAAGACTGTGGCCAGTGTTTTTCTTTTGAAT TTAGCACTGGCTGACTTATGCTTTTTACTGACTTTGCCACTATGGGCTGTCTACACAGCTATGGAATAC CGCTGGCCCTTTGGCAATTACCTATGTAAGATTGCTTCAGCCAGCGTCAGTTTCAACCTGTACGCTAGT GTGTTTCTACTCACGTGTCTCAGCATTGATCGATACCTGGCTATTGTTCACCCAATGAAGTCCCGCCTT CGACGCACAATGCTTGTAGCCAAAGTCACCTGCATCATCATTTGGCTGCTGGCAGGCTTGGCCAGTTTG CCAGCTATAATCCATCGAAATGTATTTTTCATTGAGAACACCAATATTACAGTTTGTGCTTTCCATTAT GAGTCCCAAAATTCAACCCTTCCGATAGGGCTGGGCCTGACCAAAAATATACTGGGTTTCCTGTTTCCT TTTCTGATCATTCTTACAAGTTATACTCTTATTTGGAAGGCCCTAAAGAAGGCTTATGAAATTCAGAAG AACAAACCAAGAAATGATGATATTTTTAAGATAATTATGGCAATTGTGCTTTTCTTTTTCTTTTCCTGG ATTCCCCACCAAATATTCACTTTTCTGGATGTATTGATTCAACTAGGCATCATACGTGACTGTAGAATT GCAGATATTGTGGACACGGCCATGCCTATCACCATTTGTATAGCTTATTTTAACAATTGCCTGAATCCT CTTTTTTATGGCTTTCTGGGGAAAAAATTTAAAAGATATTTTCTCCAGCTTCTAAAATATATTCCCCCA AAAGCCAAATCCCACTCAAACCTTTCAACAAAAATGAGCACGCTTTCCTACCGCCCCTCAGATAATGTA AGCTCATCCACCAAGAAGCCTGCACCATGTTTTGAGGTTGAGTGA

460(AT1R (amino acid sequence)

MILNSSTEDGIKRIQDDCPKAGRHNYIFVMIPTLYSIIFVVGIFGNSLVVIVIYFYMKLKTVASVFLLN LALADLCFLLTLPLWAVYTAMEYRWPFGNYLCKIASASVSFNLYASVFLLTCLSIDRYLAIVHPMKSRL RRTMLVAKVTCIIIWLLAGLASLPAIIHRNVFFIENTNITVCAFHYESQNSTLPIGLGLTKNILGFLFP FLIILTSYTLIWKALKKAYEIQKNKPRNDDIFKIIMAIVLFFFFSWIPHQIFTFLDVLIQLGIIRDCRI ADIVDTAMPITICIAYFNNCLNPLFYGFLGKKFKRYFLQLLKYIPPKAKSHSNLSTKMSTLSYRPSDNV SSSTKKPAPCFEVE

461(IL6-00 signal peptide (nucleotide sequence)

ATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCT GCTGCCTTCCCTGCCCCA

462(IL6-00 signal peptide (amino acid sequence)

MNSFSTSAFGPVAFSLGLLLVLPAAFPAP

463(IL 6-00L 27V-00 (nucleotide sequence)

ATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCT GCTGCCTTCCCTGCCCCAGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTAC AACCTGGACCAAGTCCTTGAGCAGGGCGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACT CCGATCCAAAGGATTGTCCTGAGCGGTGAAAACGGCCTGAAGATCGACATCCATGTCATCATCCCGTAT GAAGGTCTGAGCGGCGATCAGATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGAT CATCACTTTAAGGTGATTCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGAC TATTTCGGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACCGGGACCCTG TGGAACGGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGCGTAACC ATCAACGGAGTGACCGGCTGGCGGCTGTGCGAGCGCATTTTGGCG

SEQ ID NO:464(IL 6-00L 27V-00 (amino acid sequence)

MNSFSTSAFGPVAFSLGLLLVLPAAFPAPVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVT PIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMID YFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

SEQ ID NO:465 (Natural secretion Signal + L27V (nucleotide sequence)

atggcttactccacactgttcatcattgctctcacagccgtcgtaacacaagcctcctccacacagaaa agcaacctgacaGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTACAACCTG GACCAAGTCCTTGAGCAGGGCGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATC CAAAGGATTGTCCTGAGCGGTGAAAACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGT CTGAGCGGCGATCAGATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCAC TTTAAGGTGATTCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTC GGACGGCCGTATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACCGGGACCCTGTGGAAC GGCAACAAAATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGCGTAACCATCAAC GGAGTGACCGGCTGGCGGCTGTGCGAGCGCATTTTGGCG

466 (Natural secretion Signal + L27V (amino acid sequence)

MAYSTLFIIALTAVVTQASSTQKSNLTVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPI QRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYF GRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

467(CP 6L 27V (RIIbB) (nucleotide sequence)

Atgttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtg tccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaat gggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatc gaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcaca ctggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtg ttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctg atcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAA CGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTG CCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATAC AACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAA GTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGA TGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCAC GCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATG GAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGAT ATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagat

468(CP 6L 27V (RIIbB) (amino acid sequence)

MFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQI EKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERL INPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVY NDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAH AIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLED

469(CP 12L 27V (RIIbB) (nucleotide sequence)

ATGcagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaat ctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatc catgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtg gtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggtt acgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatc actgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctcc ctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCA AGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTG GAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATC ATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAA AGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAGTAC TTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAA TGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGGAAC ATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGA TCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcga

470(CP 12L 27V (RIIbB) (amino acid sequence)

MQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKV VYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGS LLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQI IAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVK CLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWR

471(CP 48L 27V (RIIbB) (nucleotide sequence)

ATGggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaa atgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctg cactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaa ggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatc gacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctgg cggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTT ATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGC ACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATT GAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTA GAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCA GCTTCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTG GGACCTTGCATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGA ACGAACATGGATATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagat ttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtcc agtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagc

SEQ ID NO:472(CP 48L 27V (RIIbB) (amino acid sequence)

MGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYE GIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESF IESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAV EIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYG TNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLS

473(CP 52L 27V (RIIbB) (nucleotide sequence) SEQ ID NO

ATGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGACCAAATGGGCCAGATC GAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATCCTGCACTATGGCACA CTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTATGAAGGCATCGCCGTG TTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATTATCGACGAGCGCCTG ATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAACGGAGTGACCGGCTGGCGGCTGTGCGAA CGCATTCTGGCTAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTG CCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATAC AACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAA GTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGA TGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCAC GCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATG GAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGAT ATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTATGGTCTTCACACTCGAAGATTTCGTTGGGGAC TGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTTGAACAGGGAGGTGTGTCCAGTTTGTTTCAG AATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTCCTGAGCGGTGAAAATGGG

474(CP 52L 27V (RIIbB) (amino acid sequence)

MLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAV FDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESL PFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIAR CSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMD IVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENG

SEQ ID NO:475(CP 55L 27V (RIIbB) (nucleotide sequence)

Atggacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatt tttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatc gacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggc aaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacccc gacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTG GCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTT AAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGA GAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATT ACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGG GGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGG ACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATG AAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGG TCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacag acagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggg gtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatc

SEQ ID NO:476(CP 55L 27V (RIIbB) (amino acid sequence)

MDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDG KKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFL KSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSR GQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVG SGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKI

SEQ ID NO:477(CP 83L 27V (RIIbB) (nucleotide sequence)

ATGgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccg aacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgta acagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctg ttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGA GGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTT TCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCT CAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAG GGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGA GAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAATGTTTA GCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGGAACATCGCT ACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGATCTGGA GGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaac ctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccg atccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaa ggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccct

SEQ ID NO:478(CP 83L 27V (RIIbB) (amino acid sequence)

MVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLL FRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIA QGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCL AMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYN LDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYP

479(CP 84L 27V (RIIbB) (nucleotide sequence)

atggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGT TCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCT GCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAG GGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGT AAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAG CTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCA ATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGGAACATCGCTACC TATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGATCTGGAGGT AGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtg

SEQ ID NO:480(CP 84L 27V (RIIbB) (amino acid sequence)

MDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLF RVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQ GDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLA MDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNL DQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPV

481 SEQ ID NO (CP 103L 27V (RIIbB) (nucleotide sequence)

ATGacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaag atcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggc tccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGC TCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCT TTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAA ATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATG AAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAG TACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACTGTC AAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGG AACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGT GGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacagacagcc ggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtcc gtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatc ccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtg gatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggtt

SEQ ID NO:482(CP 103L 27V (RIIbB) (amino acid sequence)

MTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILAS SSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITM KRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKR NIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVS VTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV

483(CP 120L 27V (RIIbB) (nucleotide sequence)

ATGttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgc ctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGC GAACGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCA CTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTA TACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGA GAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCT CGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCC CACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGC ATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATG GATATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggg gactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgttt cagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatc gacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaattttt aaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgac ggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtg

SEQ ID NO:484(CP 120L 27V (RIIbB) (amino acid sequence)

MFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIES LPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIA RCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNM DIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKI DIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAV

485(CP 123L 27V (RIIbB) (nucleotide sequence) SEQ ID NO

ATGaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaac cccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATT CTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTC CTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGAT GGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAA ATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCG CGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATT GGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATT ATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTA GGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcga cagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccat gtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtg taccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacg ccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggc

486(CP 123L 27V (RIIbB) (amino acid sequence)

MKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPF LKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCS RGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIV GSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIH VIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDG

487(CP 124L 27V (RIIbB) (nucleotide sequence)

ATGaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacccc gacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTG GCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTT AAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGA GAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATT ACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGG GGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGG ACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATG AAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGG TCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacag acagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggg gtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtc atcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtac cctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccg aacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaa

SEQ ID NO:488(CP 124L 27V (RIIbB) (amino acid sequence)

MKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFL KSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSR GQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVG SGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHV IIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGK

489(CP 125L 27V (RIIbB) (nucleotide sequence)

ATGatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgac ggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt AGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAA TCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAA CAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACT ATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGA CAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACT GTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAA AGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCA GGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacagaca gccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtg tccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatc atcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccct gtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaag

SEQ ID NO:490(CP 125L 27V (RIIbB) (amino acid sequence)

MITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLK SLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRG QYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGS GGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVI IPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKK

491(CP 130L 27V (RIIbB) (nucleotide sequence)

ATGaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGT TCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCT GCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAG GGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGT AAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAG CTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCA ATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGGAACATCGCTACC TATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGATCTGGAGGT AGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctg gaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatc caaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggt ctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcac tttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttc ggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacaggg

SEQ ID NO:492(CP 130L 27V (RIIbB) (amino acid sequence)

MTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFS ARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGE LALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGG SSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEG LSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTG

SEQ ID NO:493(CP 145L 27V (RIIbB) (nucleotide sequence)

ATGcccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGC ATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCA TTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAAC GATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTG AAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGC TCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCC ATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAA ATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATT GTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactgg cgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaat ctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatc catgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtg gtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggtt acgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatc actgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaac

494(CP 145L 27V (RIIbB) (amino acid sequence)

MPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYN DGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHA IGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDW RQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKV VYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLIN

495(CP 148L 27V (RIIbB) (nucleotide sequence)

ATGtccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt AGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAA TCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAA CAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACT ATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGA CAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACT GTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAA AGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCA GGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacagaca gccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtg tccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatc atcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccct gtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggc

496(CP 148L 27V (RIIbB) (amino acid sequence)

MSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGE QIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGT VKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQT AGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYP VDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDG

497(CP 157L 27V (RIIbB) (nucleotide sequence)

AtGGGAGTGACCGGCTGGCGGCTGTGCGAACGCATTCTGGCTAGCTCAAGCGGAGGTTCAGGCGGTTCC GGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAA GTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCT GATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTG GAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTA ACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAA GCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAG TTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTATG GTCTTCACACTCGAAGATTTCGTTGGGGACTGGCGACAGACAGCCGGCTACAACCTGGACCAAGTCCTT GAACAGGGAGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATTGTC CTGAGCGGTGAAAATGGGCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGCGAC CAAATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTGATC CTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCGTAT GAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACAGGGACCCTGTGGAACGGCAACAAAATT ATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGAGTAACCATCAAC

SEQ ID NO:498(CP 157L 27V (RIIbB) (amino acid sequence)

MGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSA DSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQ AFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVL EQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVI LHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN

499(SS 6L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttatt gagtcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtgataggcacc aaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattcttttttcattattgaa tctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggtgcagtagaa atcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacctcgagcagct tctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctggga ccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATggaacg aacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTttcgttggggactggcgacagaca gccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtg tccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatc atcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccct gtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

SEQ ID NO:500(SS 6L 27V (RIIbB) (amino acid sequence)

MVFTLEDSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIE SGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLG PCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGV SVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPN MIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

501(SS 12L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgaAGCgctAGCGGAGGTTCAGGCGGTTCCGGA atgtatgaaagctttattgagtcactgccattccttaaatctttggagttttctgcacgcctgaaagta gtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgat tcttttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaa gagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaact aacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagca tttgaaaggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagtta gttgccctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTcagaca gccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtg tccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatc atcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccct gtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

502(SS 12L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSAD SFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQA FERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSQTAGYNLDQVLEQGGVSSLFQNLGV SVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPN MIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

503(SS 26L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactg ccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatac aacgatggagaacaaatcattgctcagggagattcggctgattcttttttcattattgaatctggagaa gtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggtgcagtagaaatcgctcga tgctcgcggggacagtactttggagagcttgccctggtaactaacaaacctcgagcagcttctgcccac gccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctgggaccttgcatg gaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATggaacgaacatggat attgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTggtgtgtccagtttgtttcagaatctcggggtg tccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatc atcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccct gtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

504(SS 26L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVY NDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAH AIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSGVSSLFQNLGV SVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPN MIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

505(SS 45L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt AGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaa tctttggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaa caaatcattgctcagggagattcggctgattcttttttcattattgaatctggagaagtgaaaattact atgaaaagaaagggtaaatcagaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcgggga cagtactttggagagcttgccctggtaactaacaaacctcgagcagcttctgcccacgccattgggact gtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctgggaccttgcatggaaattatgaaa aggaacatcgctacctatgaagaacagttagttgccctgTATggaacgaacatggatattgtaggGTCA GGTGGATCTGGAGGtAGCTCttcTgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatc atcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccct gtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

506(SS 45L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRISASGGSGGSGMYESFIESLPFLK SLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRG QYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGS GGSGGSSSVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPN MIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

507(SS 48L 27V (RIIbB) (nucleotide sequence)

atggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgcca ttccttaaatctttggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaac gatggagaacaaatcattgctcagggagattcggctgattcttttttcattattgaatctggagaagtg aaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggtgcagtagaaatcgctcgatgc tcgcggggacagtactttggagagcttgccctggtaactaacaaacctcgagcagcttctgcccacgcc attgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctgggaccttgcatggaa attatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATggaacgaacatggatatt gtaggGTCAGGTGGATCTGGAGGtAGCTCttcTggtgaaaatgggctgaagatcgacatccatgtcatc atcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccct gtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

SEQ ID NO:508(SS 48L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSSASGGSGGSGMYESFIESLP FLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARC SRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDI VGSGGSGGSSSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPN MIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

SEQ ID NO:509(SS 52L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttatt gagtcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtgataggcacc aaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattcttttttcattattgaa tctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggtgcagtagaa atcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacctcgagcagct tctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctggga ccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATggaacg aacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTctgaagatcgacatccatgtcatc atcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccct gtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

510(SS 52L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGSASGGSGGSGMYESFI ESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVE IARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGT NMDIVGSGGSGGSSSLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPN MIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

SEQ ID NO:511(SS 55L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaa agctttattgagtcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtg ataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattcttttttc attattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggt gcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacct cgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaagg cttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctg TATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTgacatccatgtcatc atcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccct gtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

512(SS 55L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKISASGGSGGSGMYE SFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENG AVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVAL YGTNMDIVGSGGSGGSSSDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPN MIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

513(SS 83L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctAGCgctAGCGGAGGTTCAGGCGGT TCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctttggagttttctgcacgcctg aaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcg gctgattcttttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaa gtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctg gtaactaacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtg caagcatttgaaaggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaa cagttagttgccctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcT gtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

514(SS 83L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDS ADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDV QAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSVDDHHFKVILHYGTLVIDGVTPN MIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

SEQ ID NO:515(SS 84L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtgAGCgctAGCGGAGGTTCAGGC GGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctttggagttttctgcacgc ctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagat tcggctgattcttttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatca gaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgcc ctggtaactaacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggat gtgcaagcatttgaaaggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaa gaacagttagttgccctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCt tcTgatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

516(SS 84L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGD SADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMD VQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSDDHHFKVILHYGTLVIDGVTPN MIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

517(SS 85L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatAGCgctAGCGGAGGTTCA GGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctttggagttttctgca cgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgctcaggga gattcggctgattcttttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaa tcagaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagctt gccctggtaactaacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatg gatgtgcaagcatttgaaaggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctat gaagaacagttagttgccctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGC TCttcTgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

SEQ ID NO:518(SS 85L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQG DSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAM DVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSDHHFKVILHYGTLVIDGVTPN MIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

519(SS 100L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagc tttattgagtcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtgata ggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattcttttttcatt attgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggtgca gtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacctcga gcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggctt ctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTAT ggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTgacggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

520(SS 100L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVISASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVI GTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPR AASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSDGVTPN MIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

521(SS 101L 27V (RIIbB) (nucleotide sequence) SEQ ID NO

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaa agctttattgagtcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtg ataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattcttttttc attattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggt gcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacct cgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaagg cttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctg TATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTggggttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

522(SS 101L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDV IGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKP RAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSGVTPN MIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

523(SS 102L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacgggAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtat gaaagctttattgagtcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagat gtgataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattctttt ttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaat ggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaa cctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaa aggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgcc ctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTgttacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

SEQ ID NO:524(SS 102L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVD VIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNK PRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSVTPN MIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

SEQ ID NO:525(SS 103L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatg tatgaaagctttattgagtcactgccattccttaaatctttggagttttctgcacgcctgaaagtagta gatgtgataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattct tttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagag aatggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaac aaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcattt gaaaggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagtt gccctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTacgccgaac atgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

526(SS 103L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVSASGGSGGSGMYESFIESLPFLKSLEFSARLKVV DVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTN KPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSTPN MIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

527(SS 120L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgag tcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaa gtatacaacgatggagaacaaatcattgctcagggagattcggctgattcttttttcattattgaatct ggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggtgcagtagaaatc gctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacctcgagcagcttct gcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctgggacct tgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATggaacgaac atggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTttcgacggcaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

SEQ ID NO:528(SS 120L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVSASGGSGGSGMYESFIE SLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEI ARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTN MDIVGSGGSGGSSSFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

SEQ ID NO:529(SS 123L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagc tttattgagtcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtgata ggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattcttttttcatt attgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggtgca gtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacctcga gcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggctt ctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTAT ggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTaaaaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

530(SS 123L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGSASGGSGGSGMYES FIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGA VEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALY GTNMDIVGSGGSGGSSSKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

SEQ ID NO:531(SS 124L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaa agctttattgagtcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtg ataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattcttttttc attattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggt gcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacct cgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaagg cttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctg TATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTaagatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

SEQ ID NO:532(SS 124L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKSASGGSGGSGMYE SFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENG AVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVAL YGTNMDIVGSGGSGGSSSKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

533(SS 125L 27V (RIIbB) (nucleotide sequence) of SEQ ID NO:533

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtat gaaagctttattgagtcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagat gtgataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattctttt ttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaat ggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaa cctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaa aggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgcc ctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTatcactgtaaca gggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

534(SS 125L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKSASGGSGGSGMY ESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEEN GAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVA LYGTNMDIVGSGGSGGSSSITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

535(SS 130L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggAGCgctAGCGGAGGTTCAGGC GGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctttggagttttctgcacgc ctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagat tcggctgattcttttttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatca gaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgcc ctggtaactaacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggat gtgcaagcatttgaaaggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaa gaacagttagttgccctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCt tcTaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

SEQ ID NO:536(SS 130L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGSASGGSG GSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKS EVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYE EQLVALYGTNMDIVGSGGSGGSSSTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

537(SS 136L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacAGC gctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatct ttggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaa atcattgctcagggagattcggctgattcttttttcattattgaatctggagaagtgaaaattactatg aaaagaaagggtaaatcagaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacag tactttggagagcttgccctggtaactaacaaacctcgagcagcttctgcccacgccattgggactgtc aaatgtttagcaatggatgtgcaagcatttgaaaggcttctgggaccttgcatggaaattatgaaaagg aacatcgctacctatgaagaacagttagttgccctgTATggaacgaacatggatattgtaggGTCAGGT GGATCTGGAGGtAGCTCttcTaaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

SEQ ID NO:538(SS 136L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNS ASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITM KRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKR NIATYEEQLVALYGTNMDIVGSGGSGGSSSKIIDERLINPDGSLLFRVTINGVTGWRLCERILA

539(SS 143L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaa attatcgacgagcgcctgAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgag tcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaa gtatacaacgatggagaacaaatcattgctcagggagattcggctgattcttttttcattattgaatct ggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggtgcagtagaaatc gctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacctcgagcagcttct gcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctgggacct tgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATggaacgaac atggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTatcaaccccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

SEQ ID NO:540(SS 143L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIES GEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGP CMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSINPDGSLLFRVTINGVTGWRLCERILA

541(SS 145L 27V (RIIbB) (nucleotide sequence) SEQ ID NO:541

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaacAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagcttt attgagtcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtgataggc accaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattcttttttcattatt gaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggtgcagta gaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacctcgagca gcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctg ggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATgga acgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTcccgacggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

542(SS 145L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFII ESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLL GPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSPDGSLLFRVTINGVTGWRLCERILA

543(SS 147L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaa agctttattgagtcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtg ataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattcttttttc attattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggt gcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacct cgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaagg cttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctg TATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTggctccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

544(SS 147L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFF IIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFER LLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSGSLLFRVTINGVTGWRLCERILA

SEQ ID NO:545(SS 148L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggcAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtat gaaagctttattgagtcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagat gtgataggcaccaaagtatacaacgatggagaacaaatcattgctcagggagattcggctgattctttt ttcattattgaatctggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaat ggtgcagtagaaatcgctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaa cctcgagcagcttctgcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaa aggcttctgggaccttgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgcc ctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTtccctgctgttc cgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

546(SS 148L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSF FIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFE RLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSSLLFRVTINGVTGWRLCERILA

SEQ ID NO:547(SS 156L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcAGCgctAGCGGA GGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctttggagttt tctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcattgct cagggagattcggctgattcttttttcattattgaatctggagaagtgaaaattactatgaaaagaaag ggtaaatcagaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtactttgga gagcttgccctggtaactaacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgttta gcaatggatgtgcaagcatttgaaaggcttctgggaccttgcatggaaattatgaaaaggaacatcgct acctatgaagaacagttagttgccctgTATggaacgaacatggatattgtaggGTCAGGTGGATCTGGA GGtAGCTCttcTaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

548(SS 156L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTISASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIA QGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCL AMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSNGVTGWRLCERILA

SEQ ID NO:549(SS 157L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacAGCgctAGC GGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctttggag ttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatcatt gctcagggagattcggctgattcttttttcattattgaatctggagaagtgaaaattactatgaaaaga aagggtaaatcagaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtacttt ggagagcttgccctggtaactaacaaacctcgagcagcttctgcccacgccattgggactgtcaaatgt ttagcaatggatgtgcaagcatttgaaaggcttctgggaccttgcatggaaattatgaaaaggaacatc gctacctatgaagaacagttagttgccctgTATggaacgaacatggatattgtaggGTCAGGTGGATCT GGAGGtAGCTCttcTggagtgaccggctggcggctgtGCGAACGCATTCTGGCt

SEQ ID NO:550(SS 157L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQII AQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKC LAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSGVTGWRLCERILA

551(SS 158L 27V (RIIbB) (nucleotide sequence) of SEQ ID NO

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggaAGCgct AGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgagtcactgccattccttaaatctttg gagttttctgcacgcctgaaagtagtagatgtgataggcaccaaagtatacaacgatggagaacaaatc attgctcagggagattcggctgattcttttttcattattgaatctggagaagtgaaaattactatgaaa agaaagggtaaatcagaagtggaagagaatggtgcagtagaaatcgctcgatgctcgcggggacagtac tttggagagcttgccctggtaactaacaaacctcgagcagcttctgcccacgccattgggactgtcaaa tgtttagcaatggatgtgcaagcatttgaaaggcttctgggaccttgcatggaaattatgaaaaggaac atcgctacctatgaagaacagttagttgccctgTATggaacgaacatggatattgtaggGTCAGGTGGA TCTGGAGGtAGCTCttcTgtgaccggctggcggctgtGCGAACGCATTCTGGCt

552(SS 158L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGSASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQI IAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVK CLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSVTGWRLCERILA

553(SS 166L 27V (RIIbB) (nucleotide sequence)

ATGgtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgacc ggctggcggctgtGCGAAAGCgctAGCGGAGGTTCAGGCGGTTCCGGAatgtatgaaagctttattgag tcactgccattccttaaatctttggagttttctgcacgcctgaaagtagtagatgtgataggcaccaaa gtatacaacgatggagaacaaatcattgctcagggagattcggctgattcttttttcattattgaatct ggagaagtgaaaattactatgaaaagaaagggtaaatcagaagtggaagagaatggtgcagtagaaatc gctcgatgctcgcggggacagtactttggagagcttgccctggtaactaacaaacctcgagcagcttct gcccacgccattgggactgtcaaatgtttagcaatggatgtgcaagcatttgaaaggcttctgggacct tgcatggaaattatgaaaaggaacatcgctacctatgaagaacagttagttgccctgTATggaacgaac atggatattgtaggGTCAGGTGGATCTGGAGGtAGCTCttcTCGCATTCTGGCt

554(SS 166L 27V (RIIbB) (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCESASGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTK VYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAAS AHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSRILA

SEQ ID NO:555(CP 26L 27V (RIIbB) (nucleotide sequence)

ATGggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagc ggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatg ggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcac tatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggc atcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgac gagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcgg ctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATT GAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACC AAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAA TCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAA ATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCT TCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGA CCTTGCATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACG AACATGGATATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttc gttggggactggcgacagacagccggctacaacctggaccaagtccttgaacaggga

556(CP 26L 27V (RIIbB) (amino acid sequence)

MGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILH YGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWR LCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIE SGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLG PCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQG

SEQ ID NO:557(CP 45L 27V (RIIbB) (nucleotide sequence)

ATGgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagc ggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaag gtgatcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacgg ccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaac aaaattatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtg accggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTAT GAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGAT GTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTT TTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAAT GGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAA CCTCGAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAA AGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCC CTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcaca ctcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaacaggga ggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt

558(CP 45L 27V (RIIbB) (amino acid sequence)

MVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGR PYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMY ESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEEN GAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVA LYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRI

559(CP 100(+1) L27V (RIIbB) (nucleotide sequence)

ATGgacggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgac ggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaac cccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATT CTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTC CTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGAT GGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAA ATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCG CGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATT GGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATT ATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTA GGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcga cagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctc ggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccat gtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtg taccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcGAC

560(CP 100(+1) L27V (RIIbB) (amino acid sequence)

MDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERI LASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVK ITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEI MKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNL GVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVID

SEQ ID NO:561(CP 101(+1) L27V (RIIbB) (nucleotide sequence)

ATGggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggc aaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaacccc gacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTG GCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTT AAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGA GAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATT ACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGG GGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGG ACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATG AAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGG TCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacag acagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggg gtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtc atcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtac cctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacGGG

SEQ ID NO:562(CP 101(+1) L27V (RIIbB) (amino acid sequence)

MGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERIL ASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKI TMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIM KRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLG VSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDG

SEQ ID NO:563(CP 102(+1) L27V (RIIbB) (nucleotide sequence)

ATGgttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaa aagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgac ggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCt AGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAA TCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAA CAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACT ATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGA CAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACT GTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAA AGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCA GGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacagaca gccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggggtg tccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatc atcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccct gtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacgggGTT

564(CP 102(+1) L27V (RIIbB) (amino acid sequence) of SEQ ID NO:564

MVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCERILA SSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESGEVKIT MKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPCMEIMK RNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGV SVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV

565(CP 143(+1) L27V (RIIbB) (nucleotide sequence)

ATGatcaaccccgacggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGC GAACGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCA CTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTA TACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGA GAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCT CGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCC CACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGC ATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATG GATATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggg gactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgttt cagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatc gacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaattttt aaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgac ggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaa aagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgATC

SEQ ID NO 566(CP 143(+1) L27V (RIIbB) (amino acid sequence)

MINPDGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKV YNDGEQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASA HAIGTVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVG DWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIF KVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLI

567(CP 147(+1) L27V (RIIbB) (nucleotide sequence)

ATGggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGCATTCTG GCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTT AAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGA GAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATT ACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGG GGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGG ACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATG AAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGG TCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggggactggcgacag acagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgtttcagaatctcggg gtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatcgacatccatgtc atcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaatttttaaggtggtgtac cctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgacggggttacgccg aacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaaaagatcactgta acagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgacGGC

568(CP 147(+1) L27V (RIIbB) (amino acid sequence)

MGSLLFRVTINGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDG EQIIAQGDSADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIG TVKCLAMDVQAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQ TAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVY PVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDG

569(CP 156(+1) L27V (RIIbB) (nucleotide sequence)

ATGaacggagtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGT TCCGGAATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTG AAAGTAGTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCG GCTGATTCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAA GTGGAAGAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTG GTAACTAACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTG CAAGCATTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAA CAGTTAGTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCT atggtcttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtc cttgaacagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggatt gtcctgagcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggc gaccaaatgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtg atcctgcactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccg tatgaaggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaa attatcgacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcAAC

SEQ ID NO:570(CP 156(+1) L27V (RIIbB) (amino acid sequence)

MNGVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDS ADSFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDV QAFERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQV LEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKV ILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTIN

571(CP 158(+1) L27V (RIIbB) (nucleotide sequence)

ATGgtgaccggctggcggctgtGCGAACGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGA ATGTATGAAAGCTTTATTGAGTCACTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTA GTAGATGTGATAGGCACCAAAGTATACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGAT TCTTTTTTCATTATTGAATCTGGAGAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAA GAGAATGGTGCAGTAGAAATCGCTCGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACT AACAAACCTCGAGCAGCTTCTGCCCACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCA TTTGAAAGGCTTCTGGGACCTTGCATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTA GTTGCCCTGTATGGAACGAACATGGATATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtc ttcacactcgaagatttcgttggggactggcgacagacagccggctacaacctggaccaagtccttgaa cagggaggtgtgtccagtttgtttcagaatctcggggtgtccgtaactccgatccaaaggattgtcctg agcggtgaaaatgggctgaagatcgacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaa atgggccagatcgaaaaaatttttaaggtggtgtaccctgtggatgatcatcactttaaggtgatcctg cactatggcacactggtaatcgacggggttacgccgaacatgatcgactatttcggacggccgtatgaa ggcatcgccgtgttcgacggcaaaaagatcactgtaacagggaccctgtggaacggcaacaaaattatc gacgagcgcctgatcaaccccgacggctccctgctgttccgagtaaccatcaacggaGTG

572(CP 158(+1) L27V (RIIbB) (amino acid sequence)

MVTGWRLCERILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSAD SFFIIESGEVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQA FERLLGPCMEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLE QGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVIL HYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGV

573(CP 166(+1) L27V (RIIbB) (nucleotide sequence)

ATGCGCATTCTGGCtAGCTCAAGCGGAGGTTCAGGCGGTTCCGGAATGTATGAAAGCTTTATTGAGTCA CTGCCATTCCTTAAATCTTTGGAGTTTTCTGCACGCCTGAAAGTAGTAGATGTGATAGGCACCAAAGTA TACAACGATGGAGAACAAATCATTGCTCAGGGAGATTCGGCTGATTCTTTTTTCATTATTGAATCTGGA GAAGTGAAAATTACTATGAAAAGAAAGGGTAAATCAGAAGTGGAAGAGAATGGTGCAGTAGAAATCGCT CGATGCTCGCGGGGACAGTACTTTGGAGAGCTTGCCCTGGTAACTAACAAACCTCGAGCAGCTTCTGCC CACGCCATTGGGACTGTCAAATGTTTAGCAATGGATGTGCAAGCATTTGAAAGGCTTCTGGGACCTTGC ATGGAAATTATGAAAAGGAACATCGCTACCTATGAAGAACAGTTAGTTGCCCTGTATGGAACGAACATG GATATTGTAGGGTCAGGTGGATCTGGAGGTAGCTCTTCTatggtcttcacactcgaagatttcgttggg gactggcgacagacagccggctacaacctggaccaagtccttgaacagggaggtgtgtccagtttgttt cagaatctcggggtgtccgtaactccgatccaaaggattgtcctgagcggtgaaaatgggctgaagatc gacatccatgtcatcatcccgtatgaaggtctgagcggcgaccaaatgggccagatcgaaaaaattttt aaggtggtgtaccctgtggatgatcatcactttaaggtgatcctgcactatggcacactggtaatcgac ggggttacgccgaacatgatcgactatttcggacggccgtatgaaggcatcgccgtgttcgacggcaaa aagatcactgtaacagggaccctgtggaacggcaacaaaattatcgacgagcgcctgatcaaccccgac ggctccctgctgttccgagtaaccatcaacggagtgaccggctggcggctgtGCGAACGC

574(CP 166(+1) L27V (RIIbB) (amino acid sequence)

MRILASSSGGSGGSGMYESFIESLPFLKSLEFSARLKVVDVIGTKVYNDGEQIIAQGDSADSFFIIESG EVKITMKRKGKSEVEENGAVEIARCSRGQYFGELALVTNKPRAASAHAIGTVKCLAMDVQAFERLLGPC MEIMKRNIATYEEQLVALYGTNMDIVGSGGSGGSSSMVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLF QNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVID GVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGWRLCER

SEQ ID NO 575 (Joint)

GGGTCAGGTGGATCTGGAGGTAGCTCTTCT

576 (linker) of SEQ ID NO

AGCTCAAGCGGAGGTTCAGGCGGTTCCGGA

577(LC3 (nucleotide sequence))

ATGCCGTCCGAGAAGACCTTCAAACAGCGCCGGAGCTTCGAACAAAGAGTGGAAGATGTCCGGCTCATC CGGGAGCAGCACCCCACCAAGATCCCAGTGATTATAGAGCGATACAAGGGTGAGAAGCAGCTGCCCGTC CTGGACAAGACCAGTTCCTTGTACCTGATCACGTGAATATGAGCGAACTCATCAAGATAATTAGAAGGC GCCTGCAGCTCAATGCTAACCAAGCCTTCTTCCTCCTGGTGAATGGGCACAGCATGGTGAGTGTGTCCA CACCCATCTCTGAAGTGTACGAGAGCGAGAGAGATGAAGACGGCTTCCTGTACATGGTCTATGCCTCCC AGGAGACGTTCGGGACAGCACTGGCTGTGTAA

SEQ ID NO:578(LC3 (amino acid sequence))

MPSEKTFKQRRSFEQRVEDVRLIREQHPTKIPVIIERYKGEKQLPVLDKTKFLVPDHVNMSELIKIIRR RLQLNANQAFFLLVNGHSMVSVSTPISEVYESERDEDGFLYMVYASQETFGTALAV

579 (Pattern sequence)

GSAIVK

580 (model sequence) SEQ ID NO

NHGK

581 (model sequence)

SILM

582 (Pattern sequence)

AVTK

583 (model sequence)

LIVMFY

584 (Pattern sequence)

LIVMFY

SEQ ID NO:585 (Pattern sequence)

LIVM

586 SEQ ID NO (Pattern sequence)

LIVMWF

587 (Pattern sequence)

EKTQ

SEQ ID NO 588 (Pattern sequence)

LIVMAKR

589 (Pattern sequence)

LIVMAKRG

590 (model sequence)

LIVMF

591 (model sequence) of SEQ ID NO

LIVMFSYQ

SEQ ID NO:592 OgLuc L27V-LC3 fusion protein (nucleotide sequence)

ATGGTGTTTACACTCGAAGATTTCGTAGGGGACTGGCGGCAGACAGCCGGCTACAACCTGGACCAAGTC CTTGAGCAGGGCGGTGTGTCCAGTTTGTTTCAGAATCTCGGGGTGTCCGTAACTCCGATCCAAAGGATT GTCCTGAGCGGTGAAAACGGCCTGAAGATCGACATCCATGTCATCATCCCGTATGAAGGTCTGAGCGGC GATCAGATGGGCCAGATCGAAAAAATTTTTAAGGTGGTGTACCCTGTGGATGATCATCACTTTAAGGTG ATTCTGCACTATGGCACACTGGTAATCGACGGGGTTACGCCGAACATGATCGACTATTTCGGACGGCCG TATGAAGGCATCGCCGTGTTCGACGGCAAAAAGATCACTGTAACCGGGACCCTGTGGAACGGCAACAAA ATTATCGACGAGCGCCTGATCAACCCCGACGGCTCCCTGCTGTTCCGCGTAACCATCAACGGAGTGACC GGCTGGCGGCTGTGCGAGAGAATTTTGGCGGGCTCGAGCGGAGGTGGAGGTTCAGGTGGTGGCGGGAGC GGTGGAATGCCGTCCGAGAAGACCTTCAAACAGCGCCGGAGCTTCGAACAAAGAGTGGAAGATGTCCGG CTCATCCGGGAGCAGCACCCCACCAAGATCCCAGTGATTATAGAGCGATACAAGGGTGAGAAGCAGCTG CCCGTCCTGGACAAGACCAAGTTCCTTGTACCTGATCACGTGAATATGAGCGAACTCATCAAGATAATT AGAAGGCGCCTGCAGCTCAATGCTAACCAAGCCTTCTTCCTCCTGGTGAATGGGCACAGCATGGTGAGT GTGTCCACACCCATCTCTGAAGTGTACGAGAGCGAGAGAGATGAAGACGGCTTCCTGTACATGGTCTAT GCCTCCCAGGAGACGTTCGGGACAGCACTGGCTGTGTAA

593 OgLuc L27V-LC3 fusion protein (amino acid sequence)

MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSG DQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNK IIDERLINPDGSLLFRVTINGVTGWRLCERILAGSSGGGGSGGGGSGGMPSEKTFKQRRSFEQRVEDVR LIREQHPTKIPVIIERYKGEKQLPVLDKTKFLVPDHVNMSELIKIIRRRLQLNANQAFFLLVNGHSMVS VSTPISEVYESERDEDGFLYMVYASQETFGTALAV

Claims (12)

1. A light-emitting polypeptide that has a relative position to SEQ ID NO: 15 has at least one amino acid substitution selected from the group consisting of: Q18L; F1I, L27V, and V38I; F1I and L27V; F1I and V38I; L27V; M75K, L72Q, F68Y and Q18L; M75K, L72Q, F68Y, L27V, Q18L and V38I; F1I, M75K, L72Q, F68Y, L27V and V38I; F1I, M75K, L72Q, M70V and Q18L; K33N; F1I, M75K, L72Q, F68Y, K33N and V102E; and E4K, M75K, L72Q, F68Y and K33N, and

Wherein the light-emitting polypeptide has a relative sequence to SEQ ID NO: 3 have enhanced luminescence.

2. The light-emitting polypeptide of claim 1, wherein the amino acid sequence of the light-emitting polypeptide is SEQ ID NO: 19.

3. a light-emitting polypeptide that has a relative position to SEQ ID NO: 19 has at least one amino acid substitution selected from the group consisting of: Q72A; Q72G; Q72N; Q72R; Q72M; L18K; L18D; L18F; L18G; L18Y; L18W; L18H; L18R; L18M; L18N; L18P; L18S; Q72W; Q72Y; Q72F; Q72V; Q72I; Q72T; Q72N; Q72R; Q72P; Q72G; Q72A; Q72M; Q72C; Q72H; Q72S; M72F; V90R; V90Y; V90D; V90P; V90K; V90Q; K33N; K33N and 170G; K33N, T39T and 170G; K33N, G26G, M106, R112R and 170G; K33N, R11Q, T39T and 170G; K33N and T39T; K33N and K43R; K33N, T39T and K43R; K33N and Y68D; K33N, K43R and Y68D; K33N, T39T, K43R and Y68D; K33N and L27V; K33N, L27V and K43R; K33N, L27V, K43R and Y68D; K33N, L27V and Y68D; K33N, S66N and K43R; K33N, L27V, K43R and S66N; K33N, T39T and Y68D; K33N, T39T, L27V and K43R; K33N, L27V, T39T, K43R and Y68D; K33N, T39T, K43R, Y68D and 10Y; K33N, T39T, K43R, Y68D and 14S; K33N, T39T, K43R, Y68D and 16E; K33N, T39T, K43R, Y68D and 23K; K33N, T39T, K43R, Y68D and 24A; K33N, T39T, K43R, Y68D and 25L; K33N, T39T, K43R, Y68D and 27A; K33N, T39T, K43R, Y68D and 27D; K33N, T39T, K43R, Y68D and 27G; K33N, T39T, K43R, Y68D and 27I; K33N, T39T, K43R, Y68D and 27M; K33N, T39T, K43R, Y68D and 40I; K33N, T39T, K43R, Y68D and 40L; K33N, T39T, K43R, Y68D and 40Q; K33N, T39T, K43R, Y68D and 87N; K33N, T39T, K43R, Y68D and 87T; K33N, T39T, K43R, Y68D and 97E; K33N, T39T, K43R, Y68D and 100I; K33N, T39T, K43R, Y68D and 102T; K33N, T39T, K43R, Y68D and 111N; K33N, T39T, K43R, Y68D and 113K; K33N, T39T, K43R, Y68D and 125L; K33N, T39T, K43R, Y68D and 129Q; K33N, T39T, K43R, Y68D and 130K; K33N, T39T, K43R, Y68D and 142E; K33N, T39T, K43R, Y68D and 142K; K33N, T39T, K43R, Y68D and 142W; K33N, T39T, K43R, Y68D and 146G; K33N, T39T, K43R, Y68D and 147N; K33N, T39T, K43R, Y68D and 149M; K33N, T39T, K43R, Y68D and 150V; K33N, T39T, K43R, Y68D and 152E; and K33N, T39T, K43R, Y68D and 152T; and is

Wherein the light-emitting polypeptide has a relative sequence to SEQ ID NO: 3 have enhanced luminescence.

4. The light-emitting polypeptide of claim 3, wherein the amino acid sequence of the light-emitting polypeptide is SEQ ID NO: 43. 69, 89 or 93.

5. A light-emitting polypeptide that has a relative position to SEQ ID NO: 3 has Q18L, F54I, L92H and Y109F amino acid substitutions, and wherein the luminescent polypeptide has an amino acid sequence relative to SEQ ID NO: 3 have enhanced luminescence.

6. The light-emitting polypeptide of claim 5, wherein the amino acid sequence of the light-emitting polypeptide is SEQ ID NO: 5.

7. a light-emitting polypeptide that has a relative position to SEQ ID NO: 5 has at least one amino acid substitution selected from the group consisting of: L18I; V21L; G67S; F68Y; L72Q; M75K; I76F; I90T; H92R; L22F-E74K; F1I and V158I; R11Q and F109Y; L18I and V98F; V21M and M106I; F31I and V102M; Q32H and H92R; V45E and I76V; H92R and T159S; H92R and G170R; L18Q, E49D and H86R; V21M, S47P, and G51E; L46Q, M106I and L168F; G48R, I99V and I76F; D55E, H92Q and T96A; E74I, H92R, and I99T; V21M, F68L, Q69H and L142V; F68Y, L72Q, M75K and V158I; I90T and H92R; F68Y, L72Q, M75K, V158I, I90T and H92R; V21L, I76F, I90T and H92R; V21L and I76F; V21L, F68Y, L72Q, M75K and I90T; V21L, I76F and H92R; V21L, F68Y, L72Q, M75K, H92R and V158I; V21L, F68Y, L72Q, M75K, I76F, I90T, H92R, and V158I; F68Y, L72Q, M75K and H92R; V21L and H92R; F68Y, L72Q, M75K, H92R and V158I; V21L, F68Y, L72Q, M75K, I90T, H92R, and V158I; V21L, I90T, and I76F; and V21L, F68Y, L72Q, M75K, I76F and V158I; and is

Wherein the light-emitting polypeptide has a relative sequence to SEQ ID NO: 3 have enhanced luminescence.

8. The light-emitting polypeptide of claim 7, wherein the amino acid sequence of the light-emitting polypeptide is SEQ ID NO: 7.

9. a light-emitting polypeptide that has a relative position to SEQ ID NO: 7 has at least one amino acid substitution selected from the group consisting of: L22F; V36M; I76F; T128A; T128A and T154S; N135Y and R152H; L18I and P145Q; L18V and I99T; G67D and T159I; L46Q, G51R and I138S; and G51V, R112C and P113H; and is

Wherein the light-emitting polypeptide has a relative sequence to SEQ ID NO: 3 have enhanced luminescence.

10. A fusion protein comprising the light-emitting polypeptide of any one of claims 1-9 linked to a polypeptide of interest.

11. A cyclically exchanged luciferase comprising the light emitting polypeptide of any one of claims 1-9.

12. A kit comprising the light-emitting polypeptide of any one of claims 1-9, and at least one of: (a) a compound of formula (Ia) or (Ib):

wherein R is²Selected from:

or C_2-5A linear alkyl group;

R⁶selected from-H, -OH, -NH₂-OC (O) R or-OCH₂OC(O)R；

R8 is selected from

H or lower cycloalkyl;

wherein R is³And R⁴Are all H or all C_1-2An alkyl group;

w is-NH₂Halogen, -OH, -NHC (O) R, -CO₂R；

X is-S-, -O-or-NR ²²-；

Y is-H, -OH OR-OR¹¹；

Z is-CH-or-N-;

each R¹¹Independently is-C (O) R' or-CH₂OC(O)R”；

R²²Is H, CH³Or CH₂CH₃；

Each R is independently C_1-7Straight chain alkyl or C_1-7A branched alkyl group;

r' is C_1-7Straight chain alkyl or C_1-7A branched alkyl group;

the dashed bonds indicate the presence of an optional ring, which may be saturated or unsaturated;

with the proviso that when R²The method comprises the following steps:

R⁸is not provided with

With the proviso that when R²The method comprises the following steps:

R⁸is that

Or lower cycloalkyl;

and with the proviso that when R⁶Is NH₂，R²Is that

Or C_2-5An alkyl group;

or R⁸Is not provided with

And

(b) a buffering agent.

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